Method for constructing characteristic spectrum of shangtong tincture and detection method

Abstract

The present application belongs to the technical field of traditional Chinese medicine quality detection, and particularly relates to a characteristic spectrum construction method and detection method of Shangtong Tincture developed by using a new technology of ultra-high performance liquid chromatograph-diode array detector combined with multiple wavelength segment switching. The present application specifically comprises the following steps: a. Preparation of reference substance solution: taking appropriate reference substances of Bergenin, Herba Artemisiae Scopariae A, Rutin, Cinnamyl Aldehyde, Paeonol and Aconitine, dissolving the reference substances in a solvent to obtain the reference substance solution; b. Preparation of test sample solution: taking Shangtong Tincture, adding an ethanol solution to prepare the test sample solution; c. Detection: injecting the reference substance solution and the test sample solution into an ultra-high performance liquid chromatograph for detection, taking the characteristic spectrum of the reference substance solution as a reference spectrum, selecting common peaks from the characteristic spectrum of the test sample solution to construct the characteristic spectrum of Shangtong Tincture, and determining 13 common peaks; and the present application also provides a rapid and comprehensive detection method of Shangtong Tincture.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality testing technology, specifically involving a method for constructing and detecting the characteristic spectrum of typhoid fever tincture. Background Technology

[0002] The Pain-Relieving Tincture is a compound preparation composed of eight herbs: Hibiscus Leaf, Cynanchum paniculatum, Zanthoxylum nitidum, Cinnamomum cassia root, Saussurea involucrata, Menthol, Camphor, and Cinnamon Oil. It has the effects of removing blood stasis, promoting blood circulation, reducing swelling, and relieving pain. It is used for acute soft tissue injuries such as sprains, contusions, crush injuries, and tenosynovitis. This product has wide clinical applications and unique efficacy in the treatment of bruises, falls, soft tissue injuries, and rheumatic bone pain. It can improve various clinical symptoms such as pain and inflammation, and slow down the progression of the disease, making it popular among patients and medical professionals.

[0003] The existing quality standard for this product is included in the seventh volume of the "Drug Standards of the Ministry of Health" for traditional Chinese medicine preparations (standard number: WS3-B-1326-93). This standard only controls the appearance and inspection items, without qualitative or quantitative control over the ingredients in the prescription. Furthermore, Wang Yujuan, Tao Li, et al. published a method in "Chinese Pharmacist" for "Determination of the Content of Zanthoxylated Chloride, Ethoxychelonerine, Bergenin, and Bailiangjin Saponin A in Pain Relief Tincture by HPLC-DAD-ELSD". This method uses HPLC-DAD-ELSD to determine the content of four components in Pain Relief Tincture: Zanthoxylated Chloride, Ethoxychelonerine, Bergenin, and Bailiangjin Saponin A. This represents a significant improvement over the existing standard; however, this method only controls the effective components of Zanthoxylated Chloride and Cinnamomum cassia in the prescription and cannot fully and comprehensively characterize the quality characteristics and overall picture of Pain Relief Tincture.

[0004] With the upgrading of testing instruments and the cross-application of various testing technologies, comprehensive quality control of traditional Chinese medicine preparations has been provided. Therefore, it is particularly urgent and important to develop a quality control method that can simultaneously control multiple ingredients and components in the pain relief tincture, taking into account the physicochemical properties of the effective components in the prescription, the characteristics of the preparation process, and the quality attributes of the product itself. Summary of the Invention

[0005] The primary objective of this invention is to provide a method for constructing a feature map of analgesic tincture.

[0006] The method for constructing the feature map of analgesic tincture provided by the present invention includes the following steps:

[0007] a. Preparation of reference solution: Take appropriate amounts of bergenin, artemisinin, rutin, cinnamaldehyde, paeonol and aconitine reference standards, dissolve them in solvent to obtain reference solution;

[0008] b. Preparation of the test solution: Take the pain tincture and add ethanol solution to prepare the solution;

[0009] c. Detection: Inject the reference solution and the test solution into an ultra-high performance liquid chromatograph for detection. Using the characteristic chromatogram of the reference solution as a reference chromatogram, select common peaks from the characteristic chromatogram of the test solution to construct the characteristic chromatogram of the pain tincture.

[0010] Further, in step a, the method for preparing the reference solution is to accurately weigh the reference standards of bergenin, artemisinin, rutin, cinnamaldehyde, paeonol, and aconitine, and add methanol to prepare a reference solution containing 0.05 mg of bergenin, 0.02 mg of artemisinin, 0.02 mg of rutin, 1 mg of cinnamaldehyde, 0.08 mg of paeonol, and 0.02 mg of aconitine per 1 mL.

[0011] Further, in step b, the method for preparing the test solution is to accurately measure 2 mL of the pain tincture into a 10 mL volumetric flask, dilute it with an 80-90% ethanol solution and bring it to the mark, shake it well, filter it, and take the filtrate to obtain the test solution.

[0012] Further, in step c, the detection conditions of the ultra-high performance liquid chromatograph are as follows: the mobile phase is 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, using gradient elution. The gradient elution program is as follows: 0–25 min, the volume fraction of mobile phase A decreases from 97% to 50%, and the volume fraction of mobile phase B increases from 3% to 50%; 25–30 min, the volume fraction of mobile phase A decreases from 50% to 30%, and the volume fraction of mobile phase B increases from 50% to 70%; 30–30.1 min, the volume fraction of mobile phase A increases from 30% to 97%, and the volume fraction of mobile phase B decreases from 70% to 3%; 30.1–35 min, the volume fraction of mobile phase A is maintained at 97%, and the volume fraction of mobile phase B is maintained at 3%; the column temperature is 30–40℃; the detection wavelength is 190–400 nm; the flow rate is 0.3–0.75 mL / min; and the detector is a diode array detector.

[0013] Preferably, in step c, the column temperature is 35–40°C and the flow rate is 0.75 mL / min.

[0014] Furthermore, the detection wavelength is segmented and selected as follows: 0–7 min, wavelength is 210 nm; 7.01–10 min, wavelength is 220 nm; 10.01–14 min, wavelength is 270 nm; 14.01–16 min, wavelength is 337 nm; 16.01–19.5 min, wavelength is 230 nm; 19.51–35 min, wavelength is 210 nm.

[0015] The second objective of this invention is to provide a method for detecting pain tincture.

[0016] The method for detecting pain tincture provided by the present invention includes the following steps: accurately pipetting the pain tincture test solution, determining it by ultra-high performance liquid chromatography, and comparing the obtained ultra-high performance liquid chromatogram with the characteristic spectrum of pain tincture obtained by the characteristic spectrum construction method.

[0017] The ultra-high performance liquid chromatography (UHPLC) method used 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, employing gradient elution. The gradient elution program was as follows: 0–25 min, the volume fraction of mobile phase A decreased from 97% to 50%, and the volume fraction of mobile phase B increased from 3% to 50%; 25–30 min, the volume fraction of mobile phase A decreased from 50% to 30%, and the volume fraction of mobile phase B increased from 50% to 70%; 30–30.1 min, the volume fraction of mobile phase A increased from 30% to 97%, and the volume fraction of mobile phase B decreased from 70% to 3%; 30.1–35 min, maintaining the volume fraction of mobile phase A at 97% and the volume fraction of mobile phase B at 3%; the column temperature was 30–40 °C; the detection wavelength was 190–400 nm; the flow rate was 0.3–0.75 mL / min; and the detector was a diode array detector.

[0018] Preferably, the chromatographic column is an Agilent 120Aq-C18 (3.0 mm × 150 mm, 2.7 μm).

[0019] Preferably, in step c, the column temperature is 35–40°C and the flow rate is 0.75 mL / min.

[0020] Furthermore, the detection wavelength is segmented and selected as follows: 0–7 min, wavelength is 210 nm; 7.01–10 min, wavelength is 220 nm; 10.01–14 min, wavelength is 270 nm; 14.01–16 min, wavelength is 337 nm; 16.01–19.5 min, wavelength is 230 nm; 19.51–35 min, wavelength is 210 nm.

[0021] Furthermore, the preparation method of the pain tincture test solution is as follows: accurately measure 2 mL of pain tincture into a 10 mL volumetric flask, dilute with ethanol solution with a volume concentration of 80-90% and make up to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0022] The beneficial effects of this invention are:

[0023] This invention employs ultra-high performance liquid chromatography (UHPLC) with appropriate detection conditions to construct a UPL-C characteristic spectrum for pain relief tincture. It identifies 13 common peaks belonging to *Hibiscus mutabilis* leaf, *Cynanchum paniculatum*, *Zanthoxylum nitidum*, *Ardisia crenata* root, *Ardisia crenata*, camphor, and cinnamon oil. The characteristic spectrum constructed by this method fully reflects the characteristic peak information of pain relief tincture. Furthermore, the method is stable, highly precise, and reproducible, enabling rapid and effective comprehensive evaluation of the quality of pain relief tincture, providing a practical reference method for quality control and evaluation. This invention also provides a detection method for pain relief tincture. By comparing the spectrum of the test pain relief tincture with a control characteristic spectrum, the quality of the characteristic active ingredients of the pain relief tincture is detected, making the quality detection of pain relief tincture more comprehensive and ensuring its effectiveness and controllability. Attached Figure Description

[0024] Figure 1 The characteristic spectrum of the pain tincture established for this invention (showing 13 characteristic peaks, peak 2 is bergenin (Ardisia crenata), peak 3 is artemisinin (Artemisia scoparia), peak 5 is rutin (Hibiscus mutabilis leaf), peak 7 is cinnamaldehyde (cinnamon oil), peak 8 is paeonol (Cynanchum paniculatum), peak 11 is aconitine (Artemisia scoparia), peak 1 belongs to Hibiscus mutabilis leaf, peak 4 belongs to Zanthoxylum nitidum, peaks 6, 9, 10 and 13 belong to cinnamon oil, and peak 12 belongs to camphor).

[0025] Figure 2 This is a chromatogram for identifying the peaks of the test sample of the pain relief tincture using a reference standard.

[0026] Figure 3 This is a chromatogram of the organic phase of the mobile phase in Example 2.

[0027] Figure 4 This is a water phase analysis diagram of the mobile phase in Example 2.

[0028] Figure 5 This is the chromatogram of the column used in Example 2.

[0029] Figure 6 This is a graph showing the column temperature in Example 2.

[0030] Figure 7 This is a spectrum showing the detection wavelength in Example 2.

[0031] Figure 8 This is a spectrum showing the characteristic peaks of the analgesic tincture in Example 2.

[0032] Figure 9 This is the spectrum used to determine the common peaks in the characteristic spectrum of Example 2.

[0033] Figure 10 This is the precision assessment graph from Example 2.

[0034] Figure 11 This is the repeatability test spectrum in Example 2.

[0035] Figure 12 This is the stability test spectrum in Example 2. Detailed Implementation

[0036] The present invention will be further described in detail below through specific embodiments.

[0037] Example 1

[0038] 1. Instruments and reagents

[0039] 1.1 Instruments

[0040] Agilent 1290 (Agilent Technologies (China) Co., Ltd.) and Mettler Toledo XS105DU analytical balance (Mettler Toledo Instruments (Shanghai) Co., Ltd.).

[0041] 1.2 Reagents

[0042] Acetonitrile was of chromatographic grade; phosphoric acid was of analytical grade; water was Watson's distilled water; and anhydrous ethanol was of analytical grade.

[0043] 1.3 Reference Standard

[0044] Bergenin (batch number 111532-202406, purity 93.6%), paeonol (batch number 110708-202309, purity 99.9%), rutin (100080-202012, purity 91.6%), artemisinin (110895-202105, purity 98.6%), and cinnamaldehyde (110710-202404, purity 99.2%) were all purchased from the China National Institutes for Food and Drug Control. Aconitine (A2311005, purity 98.15%) was purchased from PCL.

[0045] 2. Methods and Results

[0046] 2.1 Chromatographic conditions

[0047] Column: Agilent 120Aq-C 18 (3.0mm×150mm, 2.7μm); Mobile phase: 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution according to Table 1, column temperature 35℃; flow rate 0.75mL / min, detector is diode array detector; segmented detection wavelengths are shown in Table 2; injection volume 1uL, theoretical plate number calculated based on cinnamaldehyde peak should not be less than 130000.

[0048] Table 1 shows the gradient elution conditions of the mobile phase.

[0049] Time (minutes) 0.1% Phosphoric Acid (%) Acetonitrile (%) 0～25 97→50 3→50 25～30 50→30 50→70 30～30.1 30→97 70→3 30.1～35 97 3

[0050] Table 2 shows the segmented wavelength selection.

[0051] time min wavelength nm Includes characteristic peaks 0～7 210 Peak 1, Peak 2, Peak 3 7.01～10 220 Peak 4 10.01～14 270 Peak 5, Peak 6 14.01～16 337 Peak 7 16.01～19.5 230 Peak 8, Peak 9, Peak 10, Peak 11 19.51～35 210 Peak 12, Peak 13

[0052] 2.2 Preparation of the test solution:

[0053] Accurately measure 2 mL of the pain-relieving tincture into a 10 mL volumetric flask, dilute it with 85% ethanol solution and bring it to the mark, shake well, filter, and collect the filtrate to obtain the final product.

[0054] 2.3 Preparation of reference solution

[0055] Accurately weigh the reference standards of bergenin, artemisinin, rutin, cinnamaldehyde, paeonol, and aconitine, and prepare a reference solution containing 0.05 mg bergenin, 0.02 mg artemisinin, 0.02 mg rutin, 1 mg cinnamaldehyde, 0.08 mg paeonol, and 0.02 mg aconitine per 1 mL with methanol.

[0056] 2.4 Determination Method

[0057] Accurately pipette 1 μL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatograms to obtain the characteristic chromatogram of the pain-relieving tincture. See [link to liquid chromatography]. Figure 1 and Figure 2 .

[0058] Example 2: Screening of chromatographic conditions

[0059] 2.1 Selection of mobile phase

[0060] The inventors investigated the different separation effects of organic phases (methanol, acetonitrile) and aqueous phases (water, 0.1% phosphoric acid solution, 0.1% formic acid solution).

[0061] (1) When the mobile phase is acetonitrile-0.1% phosphoric acid solution, the gradient elution program is as follows:

[0062] From 0 to 25 minutes, the volume fraction of the 0.1% phosphoric acid solution decreased from 97% to 50%, while the volume fraction of acetonitrile increased from 3% to 50%.

[0063] Over 25–30 minutes, the volume fraction of the 0.1% phosphoric acid solution decreased from 50% to 30%, while the volume fraction of acetonitrile increased from 50% to 70%.

[0064] Over 30–30.1 min, the volume fraction of the 0.1% phosphoric acid solution increased from 30% to 97%, while the volume fraction of acetonitrile decreased from 70% to 3%.

[0065] Incubate for 30.1–35 minutes, maintaining a 0.1% phosphoric acid solution volume fraction of 97% and an acetonitrile volume fraction of 3%.

[0066] (2) When the mobile phase is methanol-0.1% phosphoric acid solution, the gradient elution program is as follows:

[0067] From 0 to 25 minutes, the volume fraction of the 0.1% phosphoric acid solution decreased from 95% to 20%, while the volume fraction of methanol increased from 5% to 80%.

[0068] Over 25–30 minutes, the volume fraction of the 0.1% phosphoric acid solution decreased from 20% to 10%, while the volume fraction of methanol increased from 80% to 90%.

[0069] Over 30–30.1 min, the volume fraction of the 0.1% phosphoric acid solution increased from 10% to 95%, while the volume fraction of methanol decreased from 90% to 5%.

[0070] Incubate for 30.1–35 minutes, maintaining a 0.1% phosphoric acid solution with a volume fraction of 95% and a methanol volume fraction of 5%.

[0071] (3) When the mobile phase is acetonitrile-water, the gradient elution program is as follows:

[0072] From 0 to 25 minutes, the volume fraction of water decreased from 97% to 50%, while the volume fraction of acetonitrile increased from 3% to 50%.

[0073] Over 25–30 minutes, the volume fraction of water decreased from 50% to 30%, while the volume fraction of acetonitrile increased from 50% to 70%.

[0074] Over 30–30.1 min, the volume fraction of water increased from 30% to 97%, while the volume fraction of acetonitrile decreased from 70% to 3%.

[0075] 30.1–35 min, maintaining a water volume fraction of 97% and an acetonitrile volume fraction of 3%;

[0076] (4) When the mobile phase is acetonitrile-0.1% formic acid solution, the gradient elution program is as follows:

[0077] From 0 to 25 minutes, the volume fraction of the 0.1% formic acid solution decreased from 97% to 50%, while the volume fraction of acetonitrile increased from 3% to 50%.

[0078] Over 25–30 minutes, the volume fraction of the 0.1% formic acid solution decreased from 50% to 30%, while the volume fraction of acetonitrile increased from 50% to 70%.

[0079] Over 30–30.1 min, the volume fraction of the 0.1% formic acid solution increased from 30% to 97%, while the volume fraction of acetonitrile decreased from 70% to 3%.

[0080] Incubate for 30.1–35 minutes, maintaining a 0.1% formic acid solution volume fraction of 97% and an acetonitrile volume fraction of 3%.

[0081] See results Figure 3 , Figure 4 Ultimately, acetonitrile was chosen as the organic phase, and a 0.1% phosphoric acid solution was used as the aqueous phase for gradient elution, which resulted in better separation of characteristic peaks and a flatter baseline.

[0082] 2.2 Selection of Chromatographic Column

[0083] The inventors investigated ultra-high performance liquid chromatography (UHPLC) columns from different brands, including Agilent 120Aq-C18 (3.0 mm × 150 mm, 2.7 μm), Thermo GOLD (2.1 mm × 100 mm, 1.9 μm), and Waters HSS T3 (2.1 mm × 100 mm, 1.8 μm).

[0084] (1) An Agilent 120Aq-C18 column was used, and the gradient elution program in Table 1 of Example 1 was followed at a flow rate of 0.75 mL / min; the segmented wavelengths were:

[0085] Time (min): 0~7~7.01~10~10.01~14~14.01~16~16.01~19.5~19.51~35;

[0086] Wavelength in nm: 210→220→270→337→230→210.

[0087] (2) A Thermo Gold column (2.1 mm × 100 mm, 1.9 μm) was used with the gradient elution program in Table 1 of Example 1 at a flow rate of 0.30 mL / min. The fractional wavelengths were:

[0088] Time (min): 0~7~7.01~10~10.01~14.5~14.51~17~17.01~20~20.01~35;

[0089] Wavelength in nm: 210→220→270→337→230→210.

[0090] (3) A Waters HSS T3 column (2.1 mm × 100 mm, 1.8 μm) was used with the gradient elution program in Table 1 of Example 1 at a flow rate of 0.30 mL / min. The segmented wavelengths were:

[0091] Time (min): 0~8~8.01~10~10.01~15~15.01~18~18.01~22~22.01~35;

[0092] Wavelength in nm: 210→220→270→337→230→210.

[0093] The results showed that the Agilent 120Aq-C18 (3.0 mm × 150 mm, 2.7 μm) column provided the best separation for the sample, achieving baseline separation of all characteristic peaks. See [link to relevant documentation]. Figure 5 .

[0094] 2.3 Selection of Column Temperature

[0095] The inventors investigated column temperatures of 30℃, 35℃, and 40℃. The results showed that at these temperatures, all characteristic peaks achieved baseline separation, demonstrating good separation performance. Therefore, a column temperature between 35℃ and 40℃ is generally suitable. (See...) Figure 6 .

[0096] 2.4 Selection of detection wavelength

[0097] Using the detection conditions of Example 1 of this invention, a PDA detector is used to scan the sample across the entire wavelength range of 190nm to 400nm. Based on the principle of maximizing effective information, the wavelength that best reflects the sample composition information is selected.

[0098] The results showed that not all chromatographic peaks in the sample showed corresponding absorption at a single fixed wavelength, and cinnamaldehyde exhibited significant absorption at multiple wavelengths with extremely high response values, causing other chromatographic peaks with lower response values ​​to stand out in the same chromatogram. Therefore, appropriate wavelengths were selected based on the absorption values ​​of each chromatographic peak to maximize the number of peaks, increase absorption, provide richer information, enhance characteristic features, and improve the overall chromatogram appearance. The following segmented wavelengths were chosen:

[0099] Time (min): 0~7~7.01~10~10.01~14~14.01~16~16.01~19.5~19.51~35;

[0100] Wavelength (nm): 210→220→270→337→230→210. See Figure 7 .

[0101] 2.5 Assignment of characteristic peaks

[0102] Using the detection conditions of Example 1 of this invention, reference solutions of bergenin, artemisinin, rutin, cinnamaldehyde, paeonol, and aconitine, as well as a test solution of liniment, were injected for detection. Chromatograms were recorded, and the chromatograms of the reference standards and the test samples were compared. The chemical components in the samples were identified based on their retention times. Results: Peak 2 was bergenin (Ardisia crenata root), peak 3 was artemisinin (Artemisia scoparia), peak 5 was rutin (Hibiscus mutabilis leaf), peak 7 was cinnamaldehyde (cinnamon oil), peak 8 was paeonol (Cynanchum paniculatum), peak 11 was aconitine (Artemisia scoparia), peak 1 was attributed to Hibiscus mutabilis leaf, peak 4 to Zanthoxylum nitidum, peaks 6, 9, 10, and 13 to cinnamon oil, and peak 12 to camphor. See Figure 8 .

[0103] 2.6 Determination of common peaks in characteristic spectra

[0104] Using the detection conditions of Example 1 of this invention, 13 batches of pain-relieving tincture (batch numbers: 231101, 231103, 231106, 231201, 231205, 240302, 240304, 240501, 240502, 240504, 240603, 240702, 240705) were tested according to the method of this invention to determine the retention time of each characteristic peak. The test solution should exhibit 13 characteristic peaks. Peak 7, corresponding to the cinnamaldehyde reference standard, is peak S. The relative retention times of the remaining characteristic peaks and peak S were calculated. The retention time RSD was between 0.01% and 0.11%, and the relative retention time RSD was between 0.00% and 0.12%, indicating good batch-to-batch consistency among the 13 batches of pain-relieving tincture. The results are shown in Tables 3-6. Figure 9 .

[0105] Table 3. Retention times (min) of peaks 1-8 in 13 batches of samples.

[0106] batch number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 7(S) Peak 8 231101 1.840 5.849 6.135 8.392 10.422 11.786 15.449 16.992 231103 1.841 5.849 6.134 8.389 10.420 11.785 15.446 16.989 231106 1.839 5.846 6.134 8.387 10.420 11.782 15.444 16.986 231201 1.839 5.848 6.132 8.389 10.422 11.784 15.447 16.989 231205 1.841 5.850 6.132 8.388 10.422 11.786 15.447 16.989 240302 1.839 5.847 6.130 8.384 10.418 11.783 15.444 16.986 240304 1.840 5.850 6.131 8.387 10.424 11.786 15.449 16.990 240501 1.840 5.848 6.124 8.383 10.420 11.784 15.444 16.986 240502 1.839 5.847 6.130 8.382 10.418 11.782 15.442 16.984 240504 1.839 5.846 6.128 8.382 10.420 11.781 15.444 16.986 240603 1.840 5.849 6.123 8.384 10.423 11.785 15.446 16.989 240702 1.841 5.849 6.120 8.382 10.419 11.784 15.445 16.986 240705 1.840 5.847 6.120 8.381 10.418 11.781 15.442 16.983 RSD% 0.04 0.02 0.09 0.04 0.02 0.02 0.01 0.02

[0107] Table 4. Retention times (min) of peaks 9-13 in 13 batches of samples.

[0108] batch number Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 231101 17.887 18.379 18.838 20.083 22.346 231103 17.882 18.376 18.831 20.077 22.342 231106 17.811 18.375 18.829 20.077 22.343 231201 17.885 18.377 18.832 20.081 22.346 231205 17.884 18.377 18.823 20.074 22.343 240302 17.880 18.374 18.821 20.074 22.341 240304 17.886 18.378 18.821 20.078 22.345 240501 17.880 18.373 18.809 20.074 22.340 240502 17.880 18.372 18.812 20.073 22.341 240504 17.882 18.373 18.814 20.080 22.343 240603 17.884 18.375 18.808 20.077 22.343 240702 17.881 18.373 18.799 20.076 22.339 240705 17.879 18.371 18.804 20.074 22.338 RSD% 0.11 0.01 0.06 0.02 0.01

[0109] Table 5. Relative retention times (min) of peaks 1-8 in 13 batches of samples.

[0110] batch number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 7(S) Peak 8 231101 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 231103 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 231106 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 231201 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 231205 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 240302 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 240304 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 240501 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 240502 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 240504 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 240603 0.119 0.379 0.396 0.543 0.675 0.763 1.000 1.100 240702 0.119 0.379 0.396 0.543 0.675 0.763 1.000 1.100 240705 0.119 0.379 0.396 0.543 0.675 0.763 1.000 1.100 RSD% 0.00 0.00 0.11 0.00 0.00 0.00 0.00 0.00

[0111] Table 6. Relative retention times (min) of peaks 9-13 in 13 batches of samples.

[0112] batch number Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 231101 1.158 1.190 1.219 1.300 1.446 231103 1.158 1.190 1.219 1.300 1.446 231106 1.153 1.190 1.219 1.300 1.447 231201 1.158 1.190 1.219 1.300 1.447 231205 1.158 1.190 1.219 1.300 1.446 240302 1.158 1.190 1.219 1.300 1.447 240304 1.158 1.190 1.218 1.300 1.446 240501 1.158 1.190 1.218 1.300 1.447 240502 1.158 1.190 1.218 1.300 1.447 240504 1.158 1.190 1.218 1.300 1.447 240603 1.158 1.190 1.218 1.300 1.447 240702 1.158 1.190 1.217 1.300 1.446 240705 1.158 1.190 1.218 1.300 1.447 RSD% 0.12 0.00 0.05 0.00 0.04

[0113] Example 3 Methodological Investigation

[0114] 3.1 Precision Examination

[0115] A mixed reference solution of six substances—bergenin, artemisinin, rutin, cinnamaldehyde, paeonol, and aconitine—was injected five times consecutively, 1 μL per injection, under the detection conditions described in Example 1 of this invention. Peaks 2, 3, 5, 7, 8, and 11 corresponded to the retention times of the reference substances bergenin, artemisinin, rutin, cinnamaldehyde, paeonol, and aconitine, respectively. Peak 7, corresponding to the cinnamaldehyde reference standard, was designated as peak S. Retention times and peak areas were recorded, and the RSD values ​​for retention time and peak area were calculated. A retention time RSD between 0.00% and 0.02%, and a peak area RSD between 0.39% and 0.69%, indicated good precision. The results are shown in Tables 7 and 8. Figure 10 .

[0116] Table 7 Precision - Retention times (min) of 6 mixed control groups

[0117] serial number Peak 2 Peak 3 Peak 5 Peak 7 Peak 8 Peak 11 1 5.846 6.112 10.422 15.445 16.988 18.764 2 5.848 6.113 10.423 15.446 16.987 18.766 3 5.848 6.114 10.425 15.446 16.988 18.766 4 5.848 6.114 10.424 15.446 16.987 18.766 5 5.848 6.114 10.424 15.446 16.988 18.766 RSD% 0.02 0.01 0.01 0.00 0.00 0.00

[0118] Table 8 Precision - Peak Area of ​​6 Mixed Controls

[0119] serial number Peak 2 Peak 3 Peak 5 Peak 7 Peak 8 Peak 11 1 242.09 26.17 59.93 461.36 339.3 80.58 2 241.18 25.93 60.06 461.86 339.88 79.64 3 241.69 25.91 60.38 462.29 340.08 79.46 4 242.46 26.07 60.42 464.44 341.72 79.74 5 243.71 26.27 60.43 466.37 342.74 80.64 RSD% 0.39 0.59 0.39 0.45 0.42 0.69

[0120] 3.2 Repeatability Test

[0121] Take the pain-relieving tincture (batch number: 240501) and test it according to the method of Example 1 of this invention. Prepare 6 test solutions respectively, determine the retention time of each characteristic peak, and calculate the retention time and the RSD value of the relative retention time. The results are shown in Tables 9 to 12. Figure 11 The retention time RSD was between 0.01% and 0.09%, and the relative retention time RSD was between 0.00% and 0.1%, indicating that the method has good repeatability.

[0122] Table 9. Retention times (min) of peaks 1-8 in repeatability studies.

[0123] serial number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 7(S) Peak 8 1 1.841 5.851 6.139 8.399 10.423 11.789 15.451 16.993 2 1.840 5.849 6.136 8.395 10.419 11.785 15.447 16.989 3 1.839 5.849 6.138 8.395 10.420 11.785 15.448 16.990 4 1.840 5.852 6.138 8.396 10.424 11.789 15.451 16.993 5 1.841 5.849 6.133 8.392 10.419 11.785 15.447 16.989 6 1.840 5.849 6.135 8.392 10.422 11.786 15.449 16.992 RSD% 0.04 0.02 0.04 0.03 0.02 0.02 0.01 0.01

[0124] Table 10 Retention times (min) of peaks 9-13 in repeatability studies

[0125] serial number Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 1 17.886 18.381 18.856 20.083 22.345 2 17.883 18.337 18.854 20.082 22.344 3 17.885 18.379 18.855 20.084 22.347 4 17.887 18.381 18.845 20.083 22.348 5 17.883 18.377 18.840 20.079 22.343 6 17.887 18.379 18.838 20.083 22.346 RSD% 0.01 0.09 0.04 0.01 0.01

[0126] Table 11. Repeatability Test - Relative Retention Times (min) of Peaks 1-8

[0127] serial number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 1 0.119 0.379 0.397 0.544 0.675 0.763 1.000 1.100 2 0.119 0.379 0.397 0.543 0.674 0.763 1.000 1.100 3 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 4 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 5 0.119 0.379 0.397 0.543 0.674 0.763 1.000 1.100 6 0.119 0.379 0.397 0.543 0.675 0.763 1.000 1.100 RSD% 0.00 0.00 0.00 0.08 0.08 0.00 0.00 0.00

[0128] Table 12 Relative retention times (min) of peaks 9 and 13 in repeatability studies.

[0129] serial number Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 1 1.158 1.190 1.220 1.300 1.446 2 1.158 1.187 1.221 1.300 1.446 3 1.158 1.190 1.221 1.300 1.447 4 1.158 1.190 1.220 1.300 1.446 5 1.158 1.190 1.220 1.300 1.446 6 1.158 1.190 1.219 1.300 1.446 RSD% 0.00 0.10 0.06 0.00 0.03

[0130] 3.3 Stability Study of the Test Solution

[0131] According to the detection conditions of Example 1 of this invention, the pain-relieving tincture (batch number: 240501) was tested. The test solution was prepared according to the test solution preparation method of this invention. Its retention time and peak area were measured at 0h, 5h, 10h, 15h, 20h, 25h, 30h, and 35h, and the RSD values ​​of retention time and peak area were calculated. The RSD of retention time was between 0.02% and 0.15%, and the RSD value of peak area was between 0.45% and 2.33%, indicating that the test solution was relatively stable within 30h. The results are shown in Tables 13 to 16. Figure 12 .

[0132] Table 13 Stability Study - Retention times (min) of peaks 1-8

[0133] serial number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 7(S) Peak 8 0h 1.841 5.851 6.139 8.399 10.423 11.789 15.451 16.993 5h 1.840 5.852 6.138 8.396 10.424 11.789 15.451 16.993 10h 1.840 5.848 6.133 8.390 10.419 11.783 15.446 16.987 15h 1.840 5.849 6.135 8.392 10.422 11.786 15.449 16.992 20h 1.839 5.847 6.128 8.384 10.419 11.782 15.444 16.986 25h 1.839 5.846 6.122 8.381 10.421 11.780 15.442 16.984 30h 1.840 5.850 6.121 8.380 10.423 11.784 15.444 16.987 35h 1.841 5.848 6.114 8.377 10.418 11.780 15.439 16.981 RSD% 0.04 0.03 0.15 0.10 0.02 0.03 0.03 0.03

[0134] Table 14 Stability Study - Retention times (min) of peaks 9-13

[0135] serial number Peak 9 Peak 10 Peak 11 Peak 12 Peak 13 0h 17.886 18.381 18.856 20.083 22.345 5h 17.887 18.381 18.845 20.083 22.348 10h 17.882 18.376 18.842 20.082 22.344 15h 17.887 18.379 18.838 20.083 22.346 20h 17.882 18.374 18.823 20.081 22.344 25h 17.879 18.371 18.805 20.076 22.340 30h 17.883 18.373 18.793 20.074 22.340 35h 17.875 18.366 18.774 20.063 22.329 RSD% 0.02 0.03 0.15 0.03 0.03

[0136] Table 15 Stability Study - Peak Areas of Peaks 1-8

[0137]

[0138] Table 16 Stability Study - Peak Areas of Peaks 9-13

[0139]

[0140] Example 4

[0141] Thirteen batches of pain-relieving tincture (batch numbers: 231101, 231103, 231106, 231201, 231205, 240302, 240304, 240501, 240502, 240504, 240603, 240702, 240705) were used to prepare the test sample solution according to "2.2 Preparation of Test Solution" in Example 1. The test sample solution was accurately pipetted and injected into an ultra-high performance liquid chromatograph for analysis. The chromatographic conditions were the same as "2.1 Chromatographic Conditions" in Example 1. The detection results are shown in [Figure 1]. Figure 9 See Tables 3 to 6.

[0142] In summary, the characteristic chromatogram constructed by this invention can fully reflect the characteristic peak information of the analgesic tincture. Furthermore, the method is stable, highly precise, and reproducible, enabling rapid and effective comprehensive evaluation of the analgesic tincture's quality. The analgesic tincture detection method of this invention compares the chromatogram of the analgesic tincture product to a control characteristic chromatogram to perform quality detection of the characteristic active ingredients of the analgesic tincture, making the quality detection of the analgesic tincture more comprehensive and ensuring its effectiveness and controllability.

Claims

1. A method for constructing the characteristic spectrum of analgesic tincture, characterized in that: Includes the following steps: a. Preparation of reference solution: Take appropriate amounts of bergenin, artemisinin, rutin, cinnamaldehyde, paeonol and aconitine reference standards, dissolve them in solvent to obtain reference solution; b. Preparation of the test solution: Take the tincture of pain and add ethanol solution to prepare the test solution; the method of preparing the test solution is to accurately measure 2 mL of the tincture of pain into a 10 mL volumetric flask, dilute it with ethanol solution with a volume concentration of 80-90% and make up to the mark, shake well, filter, and take the filtrate to obtain the test solution. c. Detection: Inject the reference solution and the test solution into an ultra-high performance liquid chromatograph for detection. Using the characteristic chromatogram of the reference solution as a reference chromatogram, select common peaks from the characteristic chromatogram of the test solution to construct the characteristic chromatogram of the pain tincture. The detection conditions of the ultra-high performance liquid chromatograph are as follows: the mobile phase is 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution. The gradient elution program is as follows: 0-25 min, the volume fraction of mobile phase A decreases from 97% to 50%, and the volume fraction of mobile phase B increases from 3% to 50%; 25-30 min, the volume fraction of mobile phase A decreases from 50% to 30%, and the volume fraction of mobile phase B increases from 50% to 70%. Over 30–30.1 min, the volume fraction of mobile phase A increased from 30% to 97%, while the volume fraction of mobile phase B decreased from 70% to 3%. The column temperature was maintained at 30.1–35 min, with mobile phase A at 97% volume and mobile phase B at 3% volume; the column temperature was 30–40 °C; the detection wavelength was 190–400 nm; the flow rate was 0.3–0.75 mL / min; and the detector was a diode array detector. The detection wavelength is selected in segments: 0–7 min, wavelength is 210 nm; 7.01–10 min, wavelength is 220 nm; 10.01–14 min, wavelength is 270 nm; 14.01–16 min, wavelength is 337 nm; 16.01–19.5 min, wavelength is 230 nm; 19.51–35 min, wavelength is 210 nm.

2. The method for constructing the feature map of the analgesic tincture according to claim 1, characterized in that: In step a, the method for preparing the reference solution is to accurately weigh the reference standards of bergenin, artemisinin, rutin, cinnamaldehyde, paeonol, and aconitine, and add methanol to prepare a reference solution containing 0.05 mg of bergenin, 0.02 mg of artemisinin, 0.02 mg of rutin, 1 mg of cinnamaldehyde, 0.08 mg of paeonol, and 0.02 mg of aconitine per 1 mL.

3. The method for constructing the characteristic spectrum of the analgesic tincture according to claim 1, characterized in that: In step c, the column temperature is 35–40 °C and the flow rate is 0.75 mL / min.

4. A method for detecting pain-relieving tincture, characterized in that: The procedure includes the following steps: accurately pipetting the test solution of the pain tincture, determining it using ultra-high performance liquid chromatography (UHPLC), and comparing the obtained UHPLC chromatogram with the characteristic chromatogram of the pain tincture obtained by the characteristic chromatogram construction method according to any one of claims 1 to 3. The ultra-high performance liquid chromatography method used 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, employing gradient elution. The gradient elution program was as follows: 0–25 min, the volume fraction of mobile phase A decreased from 97% to 50%, and the volume fraction of mobile phase B increased from 3% to 50%; 25–30 min, the volume fraction of mobile phase A decreased from 50% to 30%, and the volume fraction of mobile phase B increased from 50% to 70%. Over 30–30.1 min, the volume fraction of mobile phase A increased from 30% to 97%, while the volume fraction of mobile phase B decreased from 70% to 3%. The column temperature was maintained at 30.1–35 min, with the volume fraction of mobile phase A at 97% and the volume fraction of mobile phase B at 3%; the column temperature was 30–40 °C; the detection wavelength was 190–400 nm; the flow rate was 0.3–0.75 mL / min; and the detector was a diode array detector.

5. The method for detecting pain-relieving tincture according to claim 4, characterized in that: The detection wavelength is selected in segments: 0–7 min, wavelength is 210 nm; 7.01–10 min, wavelength is 220 nm; 10.01–14 min, wavelength is 270 nm; 14.01–16 min, wavelength is 337 nm; 16.01–19.5 min, wavelength is 230 nm; 19.51–35 min, wavelength is 210 nm.

6. The method for detecting pain-relieving tincture according to claim 4, characterized in that: In step c, the column temperature is 35–40 °C and the flow rate is 0.75 mL / min.

7. The detection method according to claim 4, characterized in that: The preparation method of the pain tincture test solution is as follows: accurately measure 2 mL of pain tincture into a 10 mL volumetric flask, dilute with 80-90% ethanol solution and make up to the mark, shake well, filter, and take the filtrate to obtain the test solution.

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