UPLC (Ultra Performance Liquid Chromatography) specific chromatogram of capsule for promoting blood circulation to

Abstract

The invention provides a blood-activating and pain-relieving capsule UPLC characteristic spectrum and a construction method and application thereof, and belongs to the field of medicine analysis. The construction method comprises the following steps: preparing a test solution and a reference solution; establishing characteristic spectrums of the test sample and the reference substance by adopting an ultrahigh pressure liquid chromatograph; a chromatographic column is C18 bonded silica gel; the volume ratio of the 0.1% phosphoric acid aqueous solution to acetonitrile is 92%-10%: 8%-90%, gradient elution is carried out, and the detection wavelength is 201-230 nm; the flow velocity is 0.2 to 0.5 ml / min; the sample size is 2-10 [mu] l, and the column temperature is 35-45 DEG C; and introducing the characteristic chromatograms of different batches of test products into a traditional Chinese medicine chromatographic characteristic chromatogram evaluation system, carrying out similarity evaluation, and generating a standard characteristic chromatogram of the blood-activating and pain-relieving capsule by an average method. The method disclosed by the invention has the advantages of high separation degree, high speed, high sensitivity and good specificity, the analysis time is saved, the consumption of an organic solvent is reduced, and a reference basis is provided for ensuring safe and stable medication and controllable quality.

Description

Technical Field

[0001] The invention belongs to the technical field of drug component analysis, and particularly relates to a UPLC characteristic spectrum of a blood-activating and analgesic capsule, a construction method and an application thereof. Background Art

[0002] The characteristic map of traditional Chinese medicine is a comprehensive and quantifiable identification method. It is based on the systematic study of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, quality and stability of traditional Chinese medicine and semi-finished products of traditional Chinese medicine preparations. Traditional Chinese medicine and its preparations are multi-component complex systems. Therefore, the evaluation of their quality should adopt a detection method that is suitable for them and can provide rich identification information. The establishment of the characteristic map of traditional Chinese medicine will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then provide an overall description and evaluation of the quality of the medicine. The research and establishment of the characteristic map of traditional Chinese medicine is of great significance to improving the quality of traditional Chinese medicine and promoting the modernization of traditional Chinese medicine.

[0003] The prescription composition of Huoxue Zhitong Capsule is angelica, Panax notoginseng, vinegar frankincense, borneol, earthworm, and natural copper (calcined). It has the effects of promoting blood circulation, dispersing blood stasis, reducing swelling and relieving pain. This prescription has been used in clinical practice for many years and has good efficacy. It is one of the classic prescriptions for traumatic injuries, blood stasis, swelling and pain. It is mainly used in clinical practice to treat acute and chronic soft tissue injury diseases. Controlling its comprehensive quality information is very necessary to ensure the quality and drug safety of Huoxue Zhitong Capsule.

[0004] The current standard for Huoxue Zhitong Capsules is included in the 2015 edition of the Chinese Pharmacopoeia. This standard uses a microscope for identification and high-performance liquid chromatography for content determination. It only identifies Angelica sinensis, Panax notoginseng, and Borneol thin layer, and does not identify Rg in Panax notoginseng. 1 Reference substance, ginsenoside Rb 1 Reference substance and notoginsenoside R 1 The established testing methods and quality standards are simple and cannot reflect the overall efficacy of multi-component Chinese medicines, which seriously restricts the market expansion and clinical application of this product.

[0005] Although the scope of detection components has been further expanded in existing research, it is time-consuming. Taking the patent application "Detection Method of Fingerprint Spectrum of Huoxue Zhitong Capsules" (application publication number CNC111487351A) as an example, the detection time of this method is about 120 minutes (all peaks) based on liquid chromatography, plus the column equilibration time, the total time for detecting a sample is nearly 150 minutes; in addition, the fingerprint spectrum does not fully reflect the ingredients of Huoxue Zhitong Capsules, and the calibrated number of peaks is only 12, which cannot achieve comprehensive quality control of Huoxue Zhitong Capsules. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a UPLC characteristic spectrum of Huoxue Zhitong Capsule and a construction method and application thereof, aiming to solve at least one technical problem in the background technology.

[0007] The present invention is achieved in that:

[0008] The first aspect of the present invention provides a method for constructing a UPLC characteristic spectrum of Huoxue Zhitong Capsule, which comprises the following steps:

[0009] S1. Take the contents of Huoxue Zhitong Capsules from different batches and prepare the test solution with methanol as solvent; ferulic acid, notoginseng saponin R 1 , Ginsenoside Rg 1 , Ginsenoside Rb 1 , ginsenoside Rd, coniferyl ferulate, ligustilide, 11-carbonyl-β-acetyl-boswellic acid, elemenic acid, 3-acetyl-β-boswellic acid reference substances were prepared using methanol as solvent to prepare reference substance solutions;

[0010] S2. Pipette the test solution and the reference solution into an ultra-high pressure liquid chromatograph, record the chromatograms to establish the characteristic spectra of the test and reference substances;

[0011] The liquid chromatography conditions include: the chromatographic column is C18 bonded silica gel; the mobile phase A is 0.1% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, the volume ratio is mobile phase A: mobile phase B = 92% to 10%: 8% to 90%, gradient elution is performed, the detection wavelength is 201nm to 230nm; the flow rate is 0.2ml / min to 0.5ml / min; the injection volume is 2μl to 10μl, and the column temperature is 35°C to 45°C;

[0012] S3, exporting the characteristic spectra of different batches of test samples obtained in S2 and importing them into the evaluation system of Chinese medicine chromatographic characteristic spectra, performing similarity evaluation, and generating the standard characteristic spectra of Huoxue Zhitong Capsules by the average method;

[0013] Preferably, the construction method further comprises establishing a characteristic spectrum of a single medicinal material test sample and identifying characteristic peaks; the single medicinal material test sample refers to each medicinal material in the prescription composition of Huoxue Zhitong Capsule.

[0014] Preferably, the gradient elution procedure is specifically:

[0015] 0min~3min, mobile phase A 92%→80%, mobile phase B 8%→20%;

[0016] 3min~6min, mobile phase A 80%→75%, mobile phase B 20%→25%;

[0017] 6min~6.5min, mobile phase A 75%→73%, mobile phase B 25%→27%;

[0018] 6.5min~14min, mobile phase A 73%→62%, mobile phase B 27%→38%;

[0019] 14min~17min, mobile phase A 62%→52%, mobile phase B 38%→48%;

[0020] 17min~19min, mobile phase A 52%→50%, mobile phase B 48%→50%;

[0021] 19min~25min, mobile phase A 50%→44%, mobile phase B 50%~56%;

[0022] 25min-27min, mobile phase A 44%→30%, mobile phase B 56%→70%;

[0023] 27min~33min, mobile phase A 30%→17%, mobile phase B 70%→83%;

[0024] 33min-34min, mobile phase A 17%→10%, mobile phase B 83%→90%;

[0025] 34-35 min, mobile phase A 10%, mobile phase B 90%;

[0026] 35min~37min, mobile phase A 10%→92%, mobile phase B 90%→8%.

[0027] Preferably, the preparation steps of the test solution are: accurately weigh 2.0 g of the content of Huoxue Zhitong Capsule, place it in a 50 ml stoppered triangular conical flask, add 20 ml of 70% methanol, weigh the mass, ultrasonically treat for 30 minutes, place at room temperature, weigh the mass again, add 70% methanol to make up for the lost mass, shake well, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the Huoxue Zhitong Capsule test solution.

[0028] Preferably, the preparation steps of the reference solution are: taking ferulic acid, notoginsenoside R 1 , Ginsenoside Rg 1 , Ginsenoside Rb 1An appropriate amount of ginsenoside Rd, coniferyl ferulate, ligustilide, 11-carbonyl-β-boswellic acid, 11-carbonyl-β-acetyl-boswellic acid, elemenic acid, and 3-acetyl-β-boswellic acid reference substances are accurately weighed and 70% methanol is added to prepare reference substance solutions of different concentrations with a gradient distribution. The concentration of each component in the reference substance solution is 50μg / ml to 500μg / ml.

[0029] Take a single medicinal material of the same dosage as the prescription of Huoxue Zhitong Capsule to prepare a single medicinal material test solution, draw the single medicinal material test solution and the reference solution and inject them into the liquid chromatograph for determination, and establish the characteristic spectrum of the single medicinal material test solution;

[0030] The characteristic peak identification step is: according to the characteristic spectrum of the single medicinal material test solution, confirm the attribution of the characteristic common peaks in the characteristic spectrum, and identify 10 characteristic peaks therein by comparing with the reference substance method.

[0031] The second aspect of the present invention provides the UPLC characteristic spectrum of Huoxue Zhitong Capsule constructed by the above construction method.

[0032] Preferably, in the standard characteristic spectrum, the number of characteristic common peaks is 20; among the characteristic common peaks, peak 2, peak 3, peak 4, and peak 5 are from Panax notoginseng medicinal material, peak 1, peak 6, peak 7, peak 8, peak 9, peak 10, and peak 11 are from Angelica sinensis medicinal material, peak 14 is from Angelica sinensis medicinal material and Eupolyphaga sinensis medicinal material, and peak 12, peak 13, peak 15, peak 16, peak 17, peak 18, peak 19, and peak 20 are from vinegar frankincense medicinal material.

[0033] Preferably, peak No. 8 is selected as the S peak, and the relative retention time of each characteristic common peak and the S peak is within ±10% of the specified value, and the specified value of the retention time is set as: peak No. 1 is 0.24, peak No. 2 is 0.37, peak No. 3 is 0.40, peak No. 4 is 0.68, peak No. 5 is 0.81, peak No. 6 is 0.86, peak No. 7 is 0.89, peak No. 8 is 1.00, peak No. 9 is 1.02, peak No. 10 is 1.27, peak No. 11 is 1.43, peak No. 12 is 1.59, peak No. 13 is 1.71, peak No. 14 is 1.73, peak No. 15 is 1.76, peak No. 16 is 1.78, peak No. 17 is 1.83, peak No. 18 is 1.88, peak No. 19 is 1.91, and peak No. 20 is 1.93.

[0034] Preferably, among the 10 characteristic peaks identified, peak 1 corresponds to ferulic acid, peak 2 corresponds to notoginsenoside R 1 , Peak 3 corresponds to ginsenoside Rg 1 , Peak 4 corresponds to ginsenoside Rb 1, peak 5 corresponds to ginsenoside Rd, peak 6 corresponds to coniferyl ferulate, peak 8 corresponds to ligustilide, peak 16 corresponds to 11-carbonyl-β-acetyl-boswellic acid, peak 17 corresponds to elemenic acid, and peak 20 corresponds to 3-acetyl-β-boswellic acid.

[0035] The third aspect of the present invention provides a method for establishing the above-mentioned UPLC characteristic spectrum of Huoxue Zhitong Capsules or the application of the UPLC characteristic spectrum of Huoxue Zhitong Capsules in the field of Huoxue Zhitong Capsules detection; the application method is: the Huoxue Zhitong Capsules sample is processed according to steps S1 and S2 to obtain the characteristic spectrum of the sample, the standard characteristic spectrum obtained in step S3 is used as a control, and the characteristic spectrum similarity is calculated using a traditional Chinese medicine chromatographic characteristic spectrum evaluation system, and the sample is judged whether it is qualified according to the similarity standard, and the sample is qualified if the similarity is not less than 0.90.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention improves the liquid chromatography conditions, thereby separating more common characteristic peaks in a shorter time (elution time does not exceed 40 min), and thus establishing a more accurate and comprehensive UPLC characteristic spectrum of Huoxue Zhitong Capsules.

[0038] (2) The method for establishing the UPLC characteristic spectrum of Huoxue Zhitong Capsules and the standard characteristic spectrum thereof established by the present invention can comprehensively reflect the characteristics of Huoxue Zhitong Capsules, have high separation degree, high speed, high sensitivity, good specificity, save analysis time, save organic solvent consumption, and provide a reference basis for ensuring the safety, stability and quality control of medication.

[0039] (3) The method for establishing the UPLC characteristic spectrum of Huoxue Zhitong Capsules and the standard characteristic spectrum thereof described in the present invention can be used to detect and evaluate Huoxue Zhitong Capsules products that have been marketed in China, so as to control the quality of Huoxue Zhitong Capsules commercially available preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the reference characteristic spectrum of Huoxue Zhitong Capsule;

[0041] Figure 2 This is the full wavelength scanning image of Huoxue Zhitong Capsule;

[0042] Figure 3 This is the chromatogram of Huoxue Zhitong Capsule at different wavelengths;

[0043] Figure 4 This is a comparison chart of UPLC at different column temperatures;

[0044] Figure 5 This is a comparison chart of UPLC with different extraction methods;

[0045] Figure 6 This is a comparison chart of UPLC with different extraction solvents;

[0046] Figure 7 UPLC chromatograms of 10 batches of Huoxue Zhitong Capsules;

[0047] Figure 8 This is the UPLC comparison chromatogram of single herbal medicine and Huoxue Zhitong Capsule;

[0048] Fig. 9 This is the UPLC comparative chromatogram of the reference substance and Huoxue Zhitong Capsule. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific implementation cases described herein are only used to explain the present invention and are not used to limit the present invention.

[0050] Example 1

[0051] The method for constructing a UPLC characteristic spectrum of Huoxue Zhitong Capsule in this embodiment comprises the following steps:

[0052] 1. Determination conditions: Chromatographic conditions and system suitability test; Chromatographic column: Waters Acquity UPLC BEH C 18(2.1×100mm, 1.7μm); mobile phase: A-0.1% phosphoric acid aqueous solution, B-acetonitrile; detection wavelength: 203nm; flow rate: 0.4mL / min; injection volume: 5μl, column temperature: 45℃. Gradient elution conditions were as follows: 0 min to 3 min, mobile phase A 92% → 80%, mobile phase B 8% → 20%; 3 min to 6 min, mobile phase A 80% → 75%, mobile phase B 20% → 25%; 6 min to 6.5 min, mobile phase A 75% → 73%, mobile phase B 25% → 27%; 6.5 min to 14 min, mobile phase A 73% → 62%, mobile phase B 27% → 38%; 14 min to 17 min, mobile phase A 62% → 52%, mobile phase B 38% → 48%; 17 min to 19 min, mobile phase A 52% → 50%, mobile phase B 48% → 50%; 19 min to 25 min, mobile phase A 50% → 44%, mobile phase B 50-56%→20%; 25min~27min, mobile phase A 44%→30%, mobile phase B 56%→70%; 27min~33min, mobile phase A 30%→17%, mobile phase B 70%→83%; 33min~34min, mobile phase A 17%→10%, mobile phase B 83%→90%; 34min~35min, mobile phase A 10%, mobile phase B 90%; 35min~37min, mobile phase A 10%→92%, mobile phase B 90%→8%;

[0053] 2. Preparation of test solution: Take about 2.0g of the contents of Huoxue Zhitong Capsule, weigh accurately, place in a 50ml stoppered triangular conical bottle, add 20mL of 70% methanol, weigh the mass, ultrasonically treat for 30min, place at room temperature, weigh the mass again, add 70% methanol to make up the lost mass, shake well, filter with a 0.22μm microporous filter membrane, take the filtrate, and get the Huoxue Zhitong Capsule test solution. Take an appropriate amount of Ligusticum chuanxiong lactone reference substance, weigh accurately, add methanol to make a solution containing 1ml per 1ml, and get the reference solution.

[0054] 3. Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into ultra-high pressure liquid chromatography, and determine.

[0055] 4. Test results: The characteristic spectrum of the test sample solution was compared with the characteristic spectrum of the reference substance solution, and the index components in the characteristic spectrum of the test sample solution were assigned and located by relative retention time to obtain the characteristic spectrum of Huoxue Zhitong Capsule. Figure 1 shown.

[0056] Example 2

[0057] In this embodiment, the chromatographic conditions are optimized as follows:

[0058] (1) Establishment of detection wavelength

[0059] Method: Mobile phase A is acetonitrile, B is 0.1% phosphoric acid (V / V) solution, linear gradient elution is performed according to the established procedure; flow rate is 0.4ml / min, column temperature is 45℃, PDA monitoring, wavelength scanning range is 190nm400nm, full wavelength scanning graph is as follows Figure 2 As shown, the chromatograms at 203nm, 254nm, and 316nm were recorded. The results are as follows Figure 3 As shown. Figure 3 It can be seen that the characteristic chromatographic peaks of Huoxue Zhitong Capsules have obvious ultraviolet absorption near 200nm-330nm, the characteristic information in the wavelength range of 200nm-230nm is obviously richer than that in other wavelength ranges, and 203nm is the detection wavelength of the index components of Panax notoginseng and vinegar frankincense. Therefore, the present invention determines 203nm as the detection wavelength of the characteristic spectrum of Huoxue Zhitong Capsules.

[0060] (2) Selection of column temperature

[0061] Take the test solution of Huoxue Zhitong Capsule and inject it at 35℃, 40℃ and 45℃ column temperature under the same chromatographic conditions to investigate the effect of column temperature on the chromatogram. The results are as follows: Figure 4 As shown in the figure, with the increase of column temperature, the elution time of ligustilide was advanced. According to the detailed information of each chromatogram, it was found that when the column temperature was 45℃, the separation between the peaks was good and the chromatographic peak shape was optimal. Therefore, 45℃ was selected as the column temperature of the chromatographic condition.

[0062] Example 3

[0063] This example investigates the extraction of the test sample. First, the extraction method is investigated, and reflux extraction and ultrasonic extraction are compared. In the experiment, the peak area of ​​ligustilide and the peak area of ​​11-carbonyl-β-boswellic acid are selected as evaluation indicators. The results are shown in Tables 1 and Figure 5 ,exist Figure 5 In the figure, S1 is ultrasonic extraction and S2 is reflux extraction. Comparing the results of ultrasonic and reflux extraction, the peak area of ​​the chromatographic peak of reflux extraction is basically consistent with that of ultrasonic extraction. In addition, the ultrasonic extraction method is easy to control, simple to operate, and has good stability. Therefore, ultrasonic extraction is selected.

[0064] Table 1 Results of investigation on extraction methods

[0065]

[0066] In this experiment, the extraction solvent was investigated, and the results are shown in Table 2 and Figure 6 ,exist Figure 6In the figure, S1 is 70% methanol; S2 is 50% methanol; S3 is methanol, i.e. 100% methanol. Comparing the results of 70% methanol, 50% methanol, and methanol extraction, the peak area of ​​the chromatographic peak of ligustilide in 70% methanol is higher than that in 50% methanol and methanol extraction. The chromatographic peak information of 70% methanol extraction is more comprehensive, so 70% methanol is selected as the extraction solvent.

[0067] Table 2 Extraction solvent investigation results

[0068]

[0069] Example 4

[0070] This example is a methodological verification of the characteristic spectrum research, which specifically includes:

[0071] (1) Precision test

[0072] Take a sample of the test product, prepare a test solution according to the test solution preparation method under Example 1, inject 6 times, and record the characteristic spectrum. The retention time of the ligustilide peak (peak 8) is used as a reference. The results of the precision test are shown in Table 3. The RSD values ​​of the relative retention time and relative peak area of ​​each chromatographic peak are no more than 0.36%, which meets the requirements of the characteristic spectrum, indicating that the precision of the instrument is good.

[0073] Table 3 Precision test results of relative retention time

[0074]

[0075] (2) Repeatability test

[0076] Take sample S1, prepare 6 samples in parallel according to the test solution preparation method under Example 1, inject them separately, and record the characteristic spectrum. The retention time of the ligustilide peak (peak 8) is used as a reference. The results of the repeatability test are shown in Table 4. The RSD values ​​of the relative retention times of each chromatographic peak are no more than 0.25%, which meets the requirements of the characteristic spectrum, indicating that the method has good repeatability.

[0077] Table 4 Relative retention time repeatability test results

[0078]

[0079]

[0080] (3) Stability test

[0081] Take the same test solution under the precision test, seal it, place it at room temperature, and according to the chromatographic conditions under Example 1, sample and measure after 0h, 2h, 4h, 8h, 12h, and 24h, and record the characteristic spectrum. The retention time of the ligustilide peak (peak 8) is used as a reference. The results of the stability test are shown in Table 5. The RSD values ​​of the relative retention times of each chromatographic peak are no more than 0.73%, which meets the requirements of the characteristic spectrum, indicating that the sample is stable within 24h, and it is recommended that the sample measurement be completed within 24h.

[0082] Table 5 Stability test results of relative retention time

[0083]

[0084]

[0085] Example 5

[0086] In this example, the characteristic spectrum method of Huoxue Zhitong Capsules established in Example 1 was used to detect 10 batches of Huoxue Zhitong Capsules to obtain characteristic spectra of the test sample solution and the mixed reference solution. The batch numbers of the 10 batches of Huoxue Zhitong Capsules are shown in Table 6. The chromatograms of the characteristic spectra of different batches of test sample solutions are shown in Table 6. Figure 7 As shown, the characteristic spectrum of the obtained test sample solution was introduced into the 2012 version of the software "Chinese Medicine Chromatographic Spectrum Similarity Evaluation System" issued by the National Pharmacopoeia Committee, and the characteristic spectrum of multiple batches of Huoxue Zhitong Capsule samples was obtained, and the similarity evaluation was performed. The average method was used as the generation method of the standard characteristic spectrum to obtain the standard characteristic spectrum of Huoxue Zhitong Capsule, and the relative retention time of each common peak was calculated. The standard characteristic spectrum has a total of 20 characteristic peaks. Among them, the peak time of peak No. 8 is relatively stable and moderate, so peak No. 8 is selected as the positioning peak S peak.

[0087] The characteristic spectra obtained from 10 batches of Huoxue Zhitong Capsules were calculated, and the ratio of the retention time of each chromatographic peak to the retention time of the S peak in the same spectrum was used to obtain the relative retention time, as shown in Table 7. As shown in Table 7, the relative retention time RSD is <1%. The characteristic spectra of the 10 batches of test sample solutions were compared with the standard characteristic spectra (i.e., the control fingerprint spectra) for similarity, and the results are shown in Table 8. As shown in Table 8, the similarity of the 10 batches of samples is ≥0.90, indicating that the components of the 10 batches of Huoxue Zhitong Capsules samples are relatively stable, indicating that the characteristic spectra are representative.

[0088] Table 6 Batch numbers and serial numbers of 10 batches of Huoxue Zhitong Capsules

[0089]

[0090] Table 7 Relative retention time table of 10 batches of test solution

[0091]

[0092] Table 8 Comparison of similarity between characteristic spectra of 10 batches of test solutions and standard characteristic spectra

[0093]

[0094] Example 6

[0095] This example is the characteristic spectrum of Huoxue Zhitong Capsule and the identification of the attribution of a single medicinal material, as follows:

[0096] Preparation of single herbal medicine solution: weigh the single herbal medicine powder according to the prescription amount of Huoxue Zhitong Capsule, put it in a 50mL conical flask, add 20mL of 70% methanol, reflux in a water bath for 2 hours, take it out and cool it to room temperature, filter it, and pass the filtrate through a 0.22μm pore size microporous filter membrane to obtain the single herbal medicine test solution;

[0097] Acquisition of characteristic spectrum of single medicinal material solution: The characteristic spectrum of single medicinal material solution was obtained by UPLC method with the same chromatographic conditions as those in the characteristic spectrum detection method of Huoxue Zhitong Capsule in Example 1.

[0098] The characteristic spectrum of the mixed reference solution is obtained by using the UPLC method with the same chromatographic conditions as those in the characteristic spectrum detection method of Huoxue Zhitong Capsules in Example 1 to obtain the characteristic spectrum of the mixed reference solution.

[0099] Determination of the characteristic spectrum of Huoxue Zhitong Capsules: The UPLC method with the same chromatographic conditions as those in the detection method of the characteristic spectrum of Huoxue Zhitong Capsules in Example 1 was used to obtain the characteristic spectrum of Huoxue Zhitong Capsules.

[0100] Generation of standard characteristic spectrum: The standard characteristic spectrum of Huoxue Zhitong Capsule is obtained by the same steps as the detection method of the characteristic spectrum of Huoxue Zhitong Capsule in Example 5;

[0101] Quality inspection: Compare the characteristic spectrum of the reference solution and the single herbal solution with the standard characteristic spectrum of Huoxue Zhitong Capsules, identify the corresponding characteristic peaks of the reference solution and the single herbal sample solution in the standard spectrum of Huoxue Zhitong Capsules, and thus attribute and locate the characteristic peaks in the characteristic spectrum of the test solution;

[0102] Technical means for attributing and locating characteristic peaks: Based on the above-mentioned quality detection of the characteristic spectrum of Huoxue Zhitong Capsules, the characteristic peaks in the standard characteristic spectrum of the test solution are attributed and located through the relative retention time of the characteristic peaks and the characteristic peak DAD-UV (diode array ultraviolet absorption spectrum, range: 200nm-400nm) spectrum identification method.

[0103] Figure 8The UPLC comparison chromatograms of single medicinal materials (eurycoma sphaerocarpa, Panax notoginseng, vinegar frankincense, and Chinese angelica) and Huoxue Zhitong Capsule (preparation) are shown in Figure 1. Figure 8 It can be seen that characteristic peaks 2, 3, 4, and 5 come from Panax notoginseng, peaks 1, 6, 7, 8, 9, 10, and 11 come from Angelica sinensis, peak 14 comes from Angelica sinensis and Eupolyphaga sinensis, and peaks 12, 13, 15, 16, 17, 18, 19, and 20 come from Olibanum vinegar.

[0104] Fig. 9 The UPLC comparison chromatograms of the reference substance and Huoxue Zhitong Capsule, where S1 is blank solvent; S2 is Huoxue Zhitong Capsule; S3 is notoginsenoside R1; S4 is ginsenoside Rg1; S5 is ginsenoside Re; S6 is ginsenoside Rb1; S7 is ginsenoside Rd; S8 is ginsenoside Rh1; S9 is ferulic acid; S10 is coniferyl ferulate; S11 is ligustilide; S12 is KBA; S13 is AKBA; S14 is elemenic acid; S15 is α-BA; S16 is β-BA; S17 is α-ABA; S18 is β-ABA. Fig. 9 As shown: the standard characteristic spectrum of the Huoxue Zhitong Capsule is compared with the characteristic spectrum of the reference solution. According to the known characteristic peaks in the characteristic spectrum of the reference solution, the corresponding characteristic peaks in the standard characteristic spectrum of the Huoxue Zhitong Capsule are identified by relative retention time, and peak 1 is located to be ferulic acid and peak 2 is notoginsenoside R. 1 , Peak 3 is ginsenoside Rg 1 , Peak 4 is ginsenoside Rb 1 , peak 6 is ginsenoside Rd, peak 7 is coniferyl ferulate, peak 9 is ligustilide, peak 18 is 11-carbonyl-β-acetyl-boswellic acid, peak 19 is elemenic acid, and peak 22 is 3-acetyl-β-boswellic acid.

[0105] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a UPLC characteristic spectrum of Huoxue Zhitong Capsule, characterized in that: The construction method comprises the following steps: S1. Prepare test solution and reference solution; S2. Pipette the test solution and the reference solution into an ultra-high pressure liquid chromatograph, record the chromatograms to establish the characteristic spectra of the test and reference substances; S3, exporting the characteristic spectra of different batches of test samples obtained in S2, and then importing them into the evaluation system of Chinese medicine chromatographic characteristic spectra, performing similarity evaluation, and generating the standard characteristic spectra of Huoxue Zhitong Capsules by the average method; Among them, the liquid chromatography conditions of S2 include: the chromatographic column is C18 bonded silica gel; the mobile phase A is 0.1% phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the volume ratio is mobile phase A: mobile phase B = 92%~10%:8%~90%, with gradient elution, and the detection wavelength is 201nm~230nm; the flow rate is 0.2ml / min~0.5ml / min; the injection volume is 2μl~10μl, and the column temperature is 35℃~45℃.

2. The method for constructing the UPLC characteristic spectrum of Huoxue Zhitong Capsule according to claim 1, characterized in that: The construction method also includes establishing a characteristic spectrum of a single medicinal material test sample and identifying characteristic peaks.

3. The method for constructing the UPLC characteristic spectrum of Huoxue Zhitong Capsule according to claim 1, characterized in that: When preparing the test solution, the contents of Huoxue Zhitong Capsules from different batches are used as test samples, and methanol is used as solvent to prepare the test solution; When preparing the reference solution, ferulic acid, notoginseng saponin R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, coniferyl ferulate, ligustilide, 11-carbonyl-β-acetyl-boswellic acid, elemenic acid, and 3-acetyl-β-boswellic acid are used as reference substances, and methanol is used as solvent to prepare the reference solution.

4. The method for constructing the UPLC characteristic spectrum of Huoxue Zhitong Capsule according to claim 1, characterized in that: The gradient elution procedure is specifically as follows: 0min~3min, mobile phase A 92%→80%, mobile phase B 8%→20%; 3min~6min, mobile phase A 80%→75%, mobile phase B 20%→25%; 6min~6.5min, mobile phase A 75%→73%, mobile phase B 25%→27%; 6.5min~14min, mobile phase A 73%→62%, mobile phase B 27%→38%; 14min~17min, mobile phase A 62%→52%, mobile phase B 38%→48%; 17min~19min, mobile phase A 52%→50%, mobile phase B 48%→50%; 19min~25min, mobile phase A 50%→44%, mobile phase B 50%~56%; 25min~27min, mobile phase A 44%→30%, mobile phase B 56%→70%; 27min~33min, mobile phase A 30%→17%, mobile phase B 70%→83%; 33min-34min, mobile phase A 17%→10%, mobile phase B 83%→90%; 34-35 min, mobile phase A 10%, mobile phase B 90%; 35min~37min, mobile phase A 10%→92%, mobile phase B 90%→8%.

5. The method for constructing the UPLC characteristic spectrum of Huoxue Zhitong Capsule according to claim 2, characterized in that: The steps for establishing the characteristic spectrum of the single medicinal material test solution are as follows: taking a single medicinal material of the same dosage as the prescription of Huoxue Zhitong Capsule to prepare a single medicinal material test solution, injecting the single medicinal material test solution and the reference substance solution into a liquid chromatograph for measurement, and establishing the characteristic spectrum of the single medicinal material test solution; The characteristic peak identification step is: according to the characteristic spectrum of the single medicinal material test solution, confirm the attribution of the characteristic common peak in the characteristic spectrum, and identify the characteristic peak therein by comparing with the reference substance method.

6. The method for constructing the UPLC characteristic spectrum of Huoxue Zhitong Capsule according to claim 3, characterized in that: The preparation steps of the test solution are as follows: accurately weigh the contents of Huoxue Zhitong Capsules, add 70% methanol, ultrasonically treat, add 70% methanol to make up for the lost mass, shake well, filter through a microporous filter membrane, and take the filtrate to obtain the Huoxue Zhitong Capsules test solution; The preparation steps of the reference solution are as follows: taking appropriate amounts of ferulic acid, notoginseng saponin R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, coniferyl ferulate, ligustilide, 11-carbonyl-β-boswellic acid, 11-carbonyl-β-acetyl-boswellic acid, elemenic acid, and 3-acetyl-β-boswellic acid reference substances, accurately weighing them, and adding 70% methanol to prepare reference solutions of different concentrations with gradient distribution, wherein the concentration of each component in the reference solution is 50 μg / ml to 500 μg / ml.

7. UPLC characteristic spectrum of Huoxue Zhitong Capsule constructed by the construction method according to any one of claims 1 to 6.

8. The UPLC characteristic spectrum of Huoxue Zhitong Capsule according to claim 7, characterized in that: There are 20 common characteristic peaks in the standard characteristic spectrum.

9. The UPLC characteristic spectrum of Huoxue Zhitong Capsule according to claim 7, characterized in that: The relative retention time of each characteristic common peak and S peak is within ±10% of the specified value.

10. The method for establishing the UPLC characteristic spectrum of Huoxue Zhitong Capsules according to any one of claims 1 to 6 or the application of the UPLC characteristic spectrum of Huoxue Zhitong Capsules according to any one of claims 7 to 9 in the detection field of Huoxue Zhitong Capsules; the application method is: the Huoxue Zhitong Capsules sample is processed according to steps S1 and S2 to obtain the characteristic spectrum of the sample, the standard characteristic spectrum obtained in step S3 is used as a control, the characteristic spectrum similarity is calculated using a traditional Chinese medicine chromatographic characteristic spectrum evaluation system, and the sample is judged whether it is qualified according to the similarity standard, and the sample is qualified if the similarity is not less than 0.90.

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