Hb-AP capsule UPLC characteristic chromatogram and construction method and application thereof

Abstract

The application provides a UPLC characteristic map of blood-activating and pain-relieving capsules, a construction method and application thereof, and belongs to the field of drug analysis. The construction method comprises the following steps: preparing a test sample solution and a control sample solution; using an ultra-high pressure liquid chromatograph to establish the characteristic map of the test sample and the control sample; the chromatographic column is C18 bonded silica gel; gradient elution is performed according to the volume ratio of 0.1% phosphoric acid aqueous solution: acetonitrile = 92% to 10%: 8% to 90%, the detection wavelength is 201 to 230 nm; the flow rate is 0.2 to 0.5 ml / min; the injection amount is 2 to 10 ul, and the column temperature is 35 to 45 DEG C; the characteristic maps of different batches of test samples are introduced into a traditional Chinese medicine chromatographic characteristic map evaluation system to perform similarity evaluation, and the average method is used to generate the standard characteristic map of the blood-activating and pain-relieving capsules. The application has high separation degree, high speed and high sensitivity, has good specificity, saves analysis time and organic solvent consumption, and provides a reference basis for ensuring drug safety, stability and quality control.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical component analysis technology, and specifically relates to the UPLC characteristic spectrum of Huoxue Zhitong capsules, its construction method, and its application. Background Technology

[0002] Traditional Chinese medicine (TCM) characteristic atlases are a comprehensive and quantifiable identification method. Based on systematic research into the chemical components of TCM, they are primarily used to evaluate the authenticity, quality, and stability of raw TCM materials and semi-finished TCM preparations. TCM and its preparations are complex multi-component systems; therefore, quality evaluation should employ appropriate detection methods that provide rich identification information. Establishing TCM characteristic atlases can comprehensively reflect the types and quantities of chemical components contained in TCM and its preparations, thereby providing an overall description and evaluation of drug quality. The research and establishment of TCM characteristic atlases are of great significance for improving the quality of TCM and promoting its modernization.

[0003] The formula for Huoxue Zhitong Capsules consists of Angelica sinensis, Panax notoginseng, vinegar-processed frankincense, borneol, Eupolyphaga sinensis, and calcined natural copper. It has the effects of promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. This formula has been used clinically for many years with good efficacy and is one of the classic formulas for treating traumatic injuries, blood stasis, swelling, and pain. Clinically, it is mainly used to treat acute and chronic soft tissue injuries. Comprehensive quality control is essential to ensure the quality and safety of Huoxue Zhitong Capsules.

[0004] The current standard for Huoxue Zhitong Capsules is included in Part I of the 2015 edition of the Chinese Pharmacopoeia. This standard uses microscopy for identification and high performance liquid chromatography for content determination. It only performs thin-layer chromatography identification of Angelica sinensis, Panax notoginseng, and borneol. It establishes detection methods for Rg1 reference standard, ginsenoside Rb1 reference standard, and Panax notoginsenoside R1 in Panax notoginseng. The quality standard is simple and cannot reflect the characteristics of the overall efficacy of multiple components of traditional Chinese medicine, which seriously restricts the market expansion and clinical application of this product.

[0005] Although existing studies have expanded the range of detectable components, they are time-consuming. Taking the patent application "Detection Method of Fingerprint Spectrum of Huoxue Zhitong Capsules" (Publication No. CNC111487351A) as an example, the detection method based on liquid chromatography takes about 120 minutes (all peaks eluted). With column equilibration time, the total time for detecting one sample is nearly 150 minutes. In addition, the fingerprint spectrum does not fully reflect the components of Huoxue Zhitong Capsules. The number of peaks calibrated is only 12, which cannot achieve comprehensive quality control of Huoxue Zhitong Capsules. Summary of the Invention

[0006] Therefore, the present invention aims to provide a UPLC feature map of a blood-activating and pain-relieving capsule, its construction method and application, in order to solve at least one technical problem in the background art.

[0007] This invention is implemented as follows:

[0008] The first aspect of this invention provides a method for constructing the UPLC feature map of a blood-activating and pain-relieving capsule, which includes the following steps:

[0009] S1. Take the contents of different batches of Huoxue Zhitong capsules and prepare the test solution with methanol as solvent; prepare the reference solution with reference standards with reference standards of ferulic acid, Panax notoginseng saponin R1, Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Rd, Ferulic acid coniferyl ester, Ligusticum lactone, 11-carbonyl-β-acetyl-boswellic acid, elemol acid, and 3-acetyl-β-boswellic acid as solvent.

[0010] S2. Inject the test solution and the reference solution into the ultra-high pressure liquid chromatograph respectively, record the chromatograms and establish the characteristic chromatograms of the test sample and the reference sample;

[0011] The liquid chromatography conditions included: a C18 bonded silica column; mobile phase A being 0.1% phosphoric acid aqueous solution and mobile phase B being acetonitrile, with a volume ratio of mobile phase A:mobile phase B = 92%–10%:8%–90%, using gradient elution; detection wavelength of 201 nm–230 nm; flow rate of 0.2 ml / min–0.5 ml / min; injection volume of 2 μl–10 μl; and column temperature of 35℃–45℃.

[0012] S3. Export the characteristic chromatograms of different batches of test samples obtained in S2 and import them into the Chinese medicine chromatographic characteristic chromatogram evaluation system to conduct similarity evaluation and generate the standard characteristic chromatogram of Huoxue Zhitong capsules using the mean method.

[0013] Preferably, the construction method further includes establishing a characteristic spectrum of the single herbal sample and identifying the characteristic peaks; the single herbal sample refers to each herbal ingredient in the prescription of the blood-activating and pain-relieving capsule.

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

[0015] From 0 min to 3 min, mobile phase A changed from 92% to 80%, and mobile phase B changed from 8% to 20%.

[0016] Over 3 to 6 minutes, mobile phase A decreased from 80% to 75%, and mobile phase B decreased from 20% to 25%.

[0017] Over 6 to 6.5 minutes, mobile phase A decreased from 75% to 73%, and mobile phase B decreased from 25% to 27%.

[0018] From 6.5 min to 14 min, mobile phase A changed from 73% to 62%, and mobile phase B changed from 27% to 38%.

[0019] From 14 to 17 minutes, mobile phase A changed from 62% to 52%, and mobile phase B changed from 38% to 48%.

[0020] From 17 to 19 minutes, mobile phase A changed from 52% to 50%, and mobile phase B changed from 48% to 50%.

[0021] From 19 min to 25 min, mobile phase A increased from 50% to 44%, and mobile phase B increased from 50% to 56%.

[0022] Between 25 and 27 minutes, mobile phase A decreased from 44% to 30%, and mobile phase B decreased from 56% to 70%.

[0023] From 27 min to 33 min, the percentage of mobile phase A decreased from 30% to 17%, and the percentage of mobile phase B decreased from 70% to 83%.

[0024] From 33 to 34 minutes, mobile phase A changed from 17% to 10%, and mobile phase B changed from 83% to 90%.

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

[0026] Between 35 and 37 minutes, mobile phase A decreased from 10% to 92%, and mobile phase B decreased from 90% to 8%.

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

[0028] Preferably, the preparation steps of the reference solution are as follows: take appropriate amounts of ferulic acid, notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, ferulic acid coniferyl ester, ligustilide, 11-carbonyl-β-boswellic acid, 11-carbonyl-β-acetyl-boswellic acid, elemol acid, and 3-acetyl-β-boswellic acid reference standards, accurately weigh them, and add 70% methanol to prepare reference solutions of different concentrations with gradient distribution. The concentration of each component in the reference solution is 50 μg / ml to 500 μg / ml.

[0029] Prepare a single herb test solution by taking the same dosage of the prescription for Huoxue Zhitong Capsules. Inject the single herb test solution and the reference solution into a liquid chromatograph for determination and establish a characteristic chromatogram of the single herb test solution.

[0030] The identification steps for the characteristic peaks are as follows: based on the characteristic spectrum of the single herbal sample solution, the attribution of the common characteristic peaks in the characteristic spectrum is confirmed, and 10 characteristic peaks are identified by comparison with the reference standard method.

[0031] The second aspect of the present invention provides an UPLC feature map of the blood-activating and pain-relieving capsule constructed by the above-described construction method.

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

[0033] Preferably, peak 8 is selected as peak S, and the relative retention time of each characteristic common peak and peak S is within ±10% of a specified value. The specified retention time values ​​are set as follows: peak 1 is 0.24, peak 2 is 0.37, peak 3 is 0.40, peak 4 is 0.68, peak 5 is 0.81, peak 6 is 0.86, peak 7 is 0.89, peak 8 is 1.00, peak 9 is 1.02, peak 10 is 1.27, peak 11 is 1.43, peak 12 is 1.59, peak 13 is 1.71, peak 14 is 1.73, peak 15 is 1.76, peak 16 is 1.78, peak 17 is 1.83, peak 18 is 1.88, peak 19 is 1.91, and peak 20 is 1.93.

[0034] Preferably, among the 10 identified characteristic peaks, peak 1 corresponds to ferulic acid, peak 2 corresponds to notoginsenoside R1, peak 3 corresponds to ginsenoside Rg1, peak 4 corresponds to ginsenoside Rb1, peak 5 corresponds to ginsenoside Rd, peak 6 corresponds to ferulic acid coniferyl ester, peak 8 corresponds to ligustilide, peak 16 corresponds to 11-carbonyl-β-acetyl-boswellic acid, peak 17 corresponds to elemol acid, and peak 20 corresponds to 3-acetyl-β-boswellic acid.

[0035] The third aspect of this invention provides a method for establishing the UPLC characteristic spectrum of the above-mentioned Huoxue Zhitong capsule or the application of the UPLC characteristic spectrum of Huoxue Zhitong capsule in the field of detection of Huoxue Zhitong capsule; the method of application is as follows: the Huoxue Zhitong capsule 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 reference, the similarity of the characteristic spectrum is calculated using a traditional Chinese medicine chromatographic characteristic spectrum evaluation system, and the similarity standard is used to determine whether the sample is qualified. If the similarity is not less than 0.90, the sample is qualified.

[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 not exceeding 40 min), and thus establishing a more accurate and comprehensive UPLC characteristic spectrum of Huoxue Zhitong capsule.

[0038] (2) The method for establishing the UPLC characteristic spectrum of Huoxue Zhitong Capsules and its standard characteristic spectrum established in this invention can comprehensively reflect the characteristics of Huoxue Zhitong Capsules. It has high separation, high speed, high sensitivity, good specificity, and saves analysis time and organic solvent consumption. It provides a reference for ensuring the safety, stability and quality control of medication.

[0039] (3) The method for establishing the UPLC characteristic spectrum of the blood-activating and pain-relieving capsules described in this invention and its standard characteristic spectrum can be used to detect and evaluate the blood-activating and pain-relieving capsule products already on the market in China, so as to control the quality of commercially available blood-activating and pain-relieving capsule preparations. Attached Figure Description

[0040] Figure 1 Characteristic chromatogram of Huoxue Zhitong Capsules (for comparison);

[0041] Figure 2 This is a full-wavelength scan of the Huoxue Zhitong capsules;

[0042] Figure 3 Chromatograms of Huoxue Zhitong capsules at different wavelengths;

[0043] Figure 4 Comparison graph of UPLC at different column temperatures;

[0044] Figure 5 Comparison chart of UPLC extraction methods;

[0045] Figure 6 Comparison of UPLC images for different extraction solvents;

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

[0047] Figure 8 Comparative UPLC chromatograms of single-herb medicinal materials and blood-activating and pain-relieving capsules;

[0048] Figure 9 This is a UPLC comparison chromatogram of the reference standard and the Huoxue Zhitong capsules. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0050] Example 1

[0051] The method for constructing the UPLC feature map of a blood-activating and pain-relieving capsule according to this embodiment includes the following steps:

[0052] 1. Determination conditions: Chromatographic conditions and system suitability test; 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℃. The gradient elution conditions were as follows: 0 min–3 min, mobile phase A 92%→80%, mobile phase B 8%→20%; 3 min–6 min, mobile phase A 80%→75%, mobile phase B 20%→25%; 6 min–6.5 min, mobile phase A 75%→73%, mobile phase B 25%→27%; 6.5 min–14 min, mobile phase A 73%→62%, mobile phase B 27%→38%; 14 min–17 min, mobile phase A 62%→52%, mobile phase B 38%→48%; 17 min–19 min, mobile phase A 52%→50%, mobile phase B 48%→50%; 19 min–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: Accurately weigh approximately 2.0 g of the contents of the Huoxue Zhitong capsules and place them in a 50 ml stoppered Erlenmeyer flask. Add 20 mL of 70% methanol, weigh the solution, sonicate for 30 min, allow to stand at room temperature, weigh again, add 70% methanol to make up the lost mass, shake well, filter through a 0.22 μm microporous membrane, and collect the filtrate to obtain the Huoxue Zhitong capsule test solution. Accurately weigh an appropriate amount of ligustilide reference standard, add methanol to prepare a solution containing 1 ml per ml, thus obtaining the reference solution.

[0054] 3. Determination method: Accurately pipette 5 μl of the reference solution and the test solution into an ultra-high pressure liquid chromatograph and determine the result.

[0055] 4. Experimental Results: The characteristic chromatogram of the test sample solution was compared with that of the reference standard solution. By using relative retention time, the indicator components in the characteristic chromatogram of the test sample solution were assigned and located, thus obtaining the characteristic chromatogram of the Huoxue Zhitong Capsule. The characteristic chromatogram of the test sample determined in this embodiment is as follows: Figure 1 As shown.

[0056] Example 2

[0057] This embodiment optimizes the chromatographic conditions as follows:

[0058] (1) Establishment of detection wavelength

[0059] Method: Mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid (V / V) solution. Linear gradient elution was performed according to a predetermined program. The flow rate was 0.4 mL / min, the column temperature was 45 °C, and PDA monitoring was used. The wavelength scanning range was 190 nm to 400 nm. The full wavelength scan is shown in the figure. Figure 2 As shown, chromatograms at 203nm, 254nm, and 316nm were recorded, and the results are as follows. Figure 3 As shown. From Figure 3 As can be seen, the characteristic chromatographic peaks of Huoxue Zhitong Capsules have obvious ultraviolet absorption in the vicinity of 200nm to 330nm. The characteristic information in the wavelength range of 200nm to 230nm is significantly richer than that in other wavelength ranges. Furthermore, 203nm is the detection wavelength for the index components of Panax notoginseng and frankincense. Therefore, this invention determines 203nm as the detection wavelength for the characteristic chromatogram of Huoxue Zhitong Capsules.

[0060] (2) Selection of column temperature

[0061] The test solution of Huoxue Zhitong Capsules was injected under the same chromatographic conditions at column temperatures of 35℃, 40℃, and 45℃ to investigate the effect of column temperature on the chromatogram. The results are as follows: Figure 4 As shown, the elution time of ligustilide increases with increasing column temperature. According to the detailed information of each chromatogram, the separation between peaks is good and the peak shape is optimal when the column temperature is 45℃. Therefore, 45℃ is selected as the column temperature for chromatographic conditions.

[0062] Example 3

[0063] This embodiment investigated the extraction of the test sample. First, the extraction methods were examined, comparing reflux extraction and ultrasonic extraction. The peak areas of ligustilide and 11-carbonyl-β-boswellic acid were selected as evaluation indicators. The results are shown in Table 1 and... Figure 5 ,exist Figure 5In the diagram, S1 represents ultrasonic extraction, and S2 represents reflux extraction. Comparing the results of ultrasonic and reflux extraction, the peak areas of the chromatographic peaks from reflux extraction and ultrasonic extraction are basically the same. Furthermore, ultrasonic extraction is easier to control, simpler to operate, and has better stability; therefore, ultrasonic extraction was chosen.

[0064] Table 1 Results of the extraction method investigation

[0065]

[0066] This experiment investigated the extraction solvent, and the results are shown in Table 2 and... Figure 6 ,exist Figure 6 In the diagram, S1 is 70% methanol; S2 is 50% methanol; and S3 is 100% methanol. Comparing the results of extraction with 70% methanol, 50% methanol, and methanol, the peak area of ​​ligustilide extracted with 70% methanol is higher than that extracted with 50% methanol and methanol. The chromatographic peak information of the 70% methanol extraction is more comprehensive. Therefore, 70% methanol was chosen as the extraction solvent.

[0067] Table 2 Results of the extraction solvent investigation

[0068]

[0069] Example 4

[0070] This embodiment is a methodological validation for feature map research, specifically including:

[0071] (1) Precision test

[0072] Take the test sample and prepare the test solution according to the method described in Example 1. Inject the sample 6 times and record the characteristic chromatograms. 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 all not greater than 0.36%, which meets the requirements of the characteristic chromatogram, indicating that the instrument has good precision.

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

[0074]

[0075] (2) Repeatability test

[0076] Take sample S1 and prepare six parallel aliquots according to the test solution preparation method in Example 1. Inject each aliquot and record the characteristic chromatograms. The retention time of the ligustilide peak (peak 8) is used as a reference. The repeatability test results are shown in Table 4. The RSD values ​​of the relative retention times of each chromatographic peak are all not greater than 0.25%, which meets the requirements of the characteristic chromatogram, indicating that the method has good repeatability.

[0077] Table 4. Repeatability test results of relative retention time

[0078]

[0079]

[0080] (3) Stability test

[0081] Take the same sample solution from the precision test, seal it, and place it at room temperature. Under the chromatographic conditions described in Example 1, inject and measure the sample after 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and record the characteristic chromatograms. Use the retention time of the ligustilide peak (peak 8) as a reference. The stability test results are shown in Table 5. The RSD values ​​of the relative retention times of each chromatographic peak are all no greater than 0.73%, meeting the requirements of the characteristic chromatogram, indicating that the sample is stable within 24 h. It is recommended that the sample determination be completed within 24 h.

[0082] Table 5. Stability test results based on relative retention time

[0083]

[0084]

[0085] Example 5

[0086] In this embodiment, the characteristic chromatographic method for Huoxue Zhitong Capsules established in Example 1 was used to detect 10 batches of Huoxue Zhitong Capsules. Characteristic chromatograms of the test solution and the mixed reference solution were obtained. The batch numbers of the 10 batches of Huoxue Zhitong Capsules are shown in Table 6. The chromatograms of the characteristic chromatograms of different batches of test sample solutions are shown below. Figure 7 As shown, the characteristic chromatograms of the obtained test sample solutions were imported into the 2012 version of the "Similarity Evaluation System for Chromatographic Spectra of Traditional Chinese Medicine" software published by the National Pharmacopoeia Commission. This yielded characteristic chromatograms of multiple batches of Huoxue Zhitong Capsules samples, which were then evaluated for similarity. The average method was used to generate the standard characteristic chromatogram, resulting in the standard characteristic chromatogram of Huoxue Zhitong Capsules. The relative retention times of each common peak were calculated. The standard characteristic chromatogram contained 20 characteristic peaks. Peak 8 had a relatively stable and moderate elution time, therefore, peak 8 was selected as the positioning peak (S peak).

[0087] The relative retention times were calculated by comparing the retention times of each chromatographic peak with the retention time of the S peak in the same chromatogram using characteristic chromatograms obtained from 10 batches of Huoxue Zhitong Capsules. Table 7 shows that the relative retention time RSD is <1%. The similarity of the characteristic chromatograms of the 10 batches of test sample solutions with the standard characteristic chromatogram (i.e., the reference fingerprint chromatogram) is compared, and the results are shown in Table 8. Table 8 shows that 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, and that the characteristic chromatogram is representative.

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

[0089]

[0090] Table 7. Relative Retention Schedule of 10 Batches of Test Solution

[0091]

[0092] Table 8. Comparison of similarity between the characteristic chromatograms of 10 batches of test sample solutions and the standard characteristic chromatograms.

[0093]

[0094] Example 6

[0095] This embodiment confirms the characteristic chromatogram of the Huoxue Zhitong capsule and the attribution of its single medicinal material, as detailed below:

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

[0097] The characteristic chromatograms of single-herb solutions were obtained by UPLC under the same chromatographic conditions as the detection method for the characteristic chromatograms of Huoxue Zhitong capsules in Example 1.

[0098] The characteristic chromatogram of the mixed reference solution was obtained by UPLC under the same chromatographic conditions as the detection method for the characteristic chromatogram of Huoxue Zhitong capsules in Example 1.

[0099] Determination of the characteristic chromatogram of Huoxue Zhitong Capsules: The characteristic chromatogram of Huoxue Zhitong Capsules was obtained by UPLC under the same chromatographic conditions as in the detection method of Huoxue Zhitong Capsules in Example 1.

[0100] Generation of standard feature maps: The standard feature maps of Huoxue Zhitong capsules were obtained by using the same steps as the detection method of feature maps of Huoxue Zhitong capsules in Example 5;

[0101] Quality testing: The characteristic spectra of the reference solution and the single herbal medicine solution are compared with the standard characteristic spectra of Huoxue Zhitong Capsules. The corresponding characteristic peaks of the reference solution and the single herbal medicine sample solution in the standard spectra of Huoxue Zhitong Capsules are identified, thereby assigning and locating the characteristic peaks in the characteristic spectra of the test solution.

[0102] Characteristic peak attribution and localization techniques: Based on the quality detection of the characteristic spectrum of the above-mentioned blood-activating and pain-relieving capsules, the characteristic peaks in the standard characteristic spectrum of the test solution are assigned and located by the relative retention time of the characteristic peaks and the characteristic peak DAD-UV (diode array ultraviolet absorption spectrum, range: 200nm-400nm) spectral identification method.

[0103] Figure 8 The UPLC comparative chromatograms are shown for single medicinal materials (Eupolyphaga sinensis, Panax notoginseng, frankincense, and Angelica sinensis) and Huoxue Zhitong capsules (preparation). 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 frankincense.

[0104] Figure 9 This is a UPLC comparison chromatogram of the reference standard and Huoxue Zhitong capsules, where S1 is blank solvent; S2 is Huoxue Zhitong capsules; S3 is Panax notoginseng saponin 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 ferulic acid coniferyl ester; S11 is ligustilide; S12 is KBA; S13 is AKBA; S14 is elemol acid; S15 is α-BA; S16 is β-BA; S17 is α-ABA; and S18 is β-ABA. Figure 9 As shown: By comparing the standard characteristic spectrum of the above-mentioned Huoxue Zhitong Capsules with the characteristic spectrum of the reference solution, and based on the known characteristic peaks in the characteristic spectrum of the reference solution, the corresponding characteristic peaks in the standard characteristic spectrum of Huoxue Zhitong Capsules were identified by relative retention time. Peak 1 was determined to be ferulic acid, peak 2 to be notoginsenoside R1, peak 3 to be ginsenoside Rg1, peak 4 to be ginsenoside Rb1, peak 6 to be ginsenoside Rd, peak 7 to be ferulic acid coniferyl ester, peak 9 to be ligustilide, peak 18 to be 11-carbonyl-β-acetyl-boswellic acid, peak 19 to be elemol acid, and peak 22 to be 3-acetyl-β-boswellic acid.

[0105] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for constructing the UPLC feature map of a blood-activating and pain-relieving capsule, characterized in that, The construction method includes the following steps: S1. Preparation of the test solution and the reference solution; the test solution is prepared using contents of different batches of Huoxue Zhitong Capsules as the test sample. The preparation steps of the test solution are as follows: accurately weigh the contents of Huoxue Zhitong Capsules, add 70% methanol, sonicate, add 70% methanol to make up the lost mass, shake well, filter through a microporous membrane, and take the filtrate to obtain the Huoxue Zhitong Capsule test solution; the preparation steps of the reference solution are as follows: take ferulic acid... Appropriate amounts of the following reference standards were accurately weighed and added to 70% methanol to prepare reference solutions of different concentrations: Panax notoginseng saponin R1, Ginsenoside Rg1, Ginsenoside Rb1, Ginsenoside Rd, Ferulic acid coniferyl ester, Ligusticolone, 11-carbonyl-β-boswellic acid, 11-carbonyl-β-acetyl-boswellic acid, elemol acid, and 3-acetyl-β-boswellic acid. S2. Inject the test solution and the reference solution into the ultra-high pressure liquid chromatograph respectively, record the chromatograms and establish the characteristic chromatograms of the test sample and the reference sample; S3. Export the characteristic chromatograms of different batches of test samples obtained in S2, and then import them into the Chinese medicine chromatographic characteristic chromatogram evaluation system to evaluate similarity and generate the standard characteristic chromatogram of Huoxue Zhitong Capsules using the mean method. The liquid chromatography conditions for S2 include: a C18 bonded silica column; mobile phase A is 0.1% phosphoric acid aqueous solution, and mobile phase B is acetonitrile, with a volume ratio of mobile phase A:mobile phase B = 92%–10%:8%–90%, using gradient elution; detection wavelength is 201 nm–230 nm; flow rate is 0.2 ml / min–0.5 ml / min; injection volume is 2 μl–10 μl; and column temperature is 35℃–45℃. The gradient elution procedure is as follows: From 0 min to 3 min, mobile phase A changed from 92% to 80%, and mobile phase B changed from 8% to 20%. From 3 to 6 minutes, mobile phase A changed from 80% to 75%, and mobile phase B changed from 20% to 25%. Over 6 to 6.5 minutes, mobile phase A decreased from 75% to 73%, and mobile phase B decreased from 25% to 27%. From 6.5 min to 14 min, mobile phase A changed from 73% to 62%, and mobile phase B changed from 27% to 38%. From 14 to 17 minutes, mobile phase A changed from 62% to 52%, and mobile phase B changed from 38% to 48%. From 17 to 19 minutes, mobile phase A changed from 52% to 50%, and mobile phase B changed from 48% to 50%. From 19 to 25 minutes, mobile phase A increased from 50% to 44%, and mobile phase B increased from 50% to 56%. Between 25 and 27 minutes, mobile phase A decreased from 44% to 30%, and mobile phase B decreased from 56% to 70%. Between 27 and 33 minutes, mobile phase A decreased from 30% to 17%, and mobile phase B decreased from 70% to 83%. Between 33 and 34 minutes, mobile phase A decreased from 17% to 10%, and mobile phase B decreased from 83% to 90%. 34-35 min, mobile phase A 10%, mobile phase B 90%; Between 35 and 37 minutes, mobile phase A decreased from 10% to 92%, and mobile phase B decreased from 90% to 8%.

2. The method for constructing the UPLC feature map of the blood-activating and pain-relieving capsule according to claim 1, characterized in that, The construction method also includes establishing characteristic spectra of single medicinal materials and identifying characteristic peaks.

3. The method for constructing the UPLC feature map of the blood-activating and pain-relieving capsule according to claim 2, characterized in that, The steps for establishing the characteristic chromatogram of the single herb test solution are as follows: take the same dose of single herb as the prescription of Huoxue Zhitong Capsule to prepare a single herb test solution, inject the single herb test solution and the reference solution into the liquid chromatograph for determination, and establish the characteristic chromatogram of the single herb test solution. The identification steps for the characteristic peaks are as follows: based on the characteristic spectrum of the single herbal sample solution, the attribution of the common characteristic peaks in the characteristic spectrum is confirmed, and the characteristic peaks are identified by comparison with the reference standard method.

4. The method for constructing the UPLC feature map of the blood-activating and pain-relieving capsule according to claim 1, characterized in that, The standard feature spectrum contains 20 characteristic peaks.

5. The method for constructing the UPLC feature map of the blood-activating and pain-relieving capsule according to claim 1, characterized in that, The relative retention times of the common peaks and the S peak are within ±10% of the specified values.

6. The application of the method for constructing the UPLC feature map of the Huoxue Zhitong capsule as described in any one of claims 1 to 5 in the field of Huoxue Zhitong capsule detection; characterized in that, The method of application is as follows: the blood-activating and pain-relieving capsule 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 reference. The similarity of the characteristic spectrum is calculated using the traditional Chinese medicine chromatographic characteristic spectrum evaluation system. The similarity standard is used to judge whether the sample is qualified. If the similarity is not less than 0.90, the sample is qualified.

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