Characteristic spectrum construction method and detection method of pain tincture

Abstract

The invention belongs to the technical field of traditional Chinese medicine quality detection, particularly relates to a new technology of combining an ultra-high performance liquid chromatograph-diode array detector with multi-time wavelength sectional switching, and develops a specific chromatogram construction method and a detection method of pain tincture. The method specifically comprises the following steps: a, preparation of a reference substance solution: taking a proper amount of bergenin, brachypekinin A, rutin, cinnamaldehyde, paeonol and aconitine reference substances, and adding a solvent for dissolving to obtain the reference substance solution; b, preparation of a test solution: taking the pain tincture, and adding an ethanol solution to prepare the test solution; c, detection: injecting the reference solution and the test solution into an ultra-high performance liquid chromatograph for detection, taking the specific chromatogram of the reference solution as a reference chromatogram, selecting common peaks from the specific chromatogram of the test solution, constructing the specific chromatogram of the pain-relieving tincture, and determining 13 common peaks; meanwhile, the invention also provides a rapid and comprehensive detection method of the pain tincture.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a characteristic spectrum construction method and a detection method of a pain tincture. Background Art

[0002] The pain tincture is a compound preparation composed of eight herbs: Hibiscus leaf, Cynanchum chinense, Zanthoxylum bungeana, Cinnabar root, Artemisia scoparia, Menthol, Camphor, and Cinnamon oil. It has the effects of removing blood stasis, activating blood circulation, reducing swelling and relieving pain. It is used for acute soft tissue injuries such as sprains, contusions, crush injuries, and tenosynovitis. This variety is widely used in clinical practice and has unique efficacy in the treatment of traumatic injuries, falls, soft tissue injuries, rheumatic bone pain, etc. It can not only improve the patient's pain, inflammation and other clinical symptoms, but also alleviate the course of the disease. It is deeply loved by patients and medical workers.

[0003] The existing quality standard of this product is included in the seventh volume of Chinese medicine formula preparations of the "Ministry of Health Drug Standards" (standard number: WS3-B-1326-93). The standard only controls the properties and inspection items, and does not conduct qualitative or quantitative control of the prescription taste. In addition, Wang Yujuan, Tao Li, etc. published the method of "HPLC-DAD-ELSD method for determining the content of nitidine chloride, ethoxychelerythrine, bergenin and 100-liangjin saponin A in the pain tincture" in "Chinese Pharmacist". This method uses HPLC-DAD-ELSD method to determine the content of nitidine chloride, ethoxychelerythrine, bergenin and 100-liangjin saponin A in the pain tincture, which has obvious progress and improvement compared with the existing standards; however, this method only controls the effective ingredients of the two medicinal flavors of nitidine and cinnabar root in the prescription, and cannot fully and comprehensively characterize the quality characteristics and overall picture of the pain tincture.

[0004] With the upgrading of detection instruments and the cross-combination of multiple detection technologies, technical support has been provided for the comprehensive quality control of Chinese patent medicines. Therefore, it is particularly urgent and important to develop a quality control method that can simultaneously control multiple medicinal flavors and multiple ingredients in the prescription of the pain tincture, combining the physical and chemical properties of the active ingredients in the prescription of the pain tincture, the characteristics of the preparation process and the quality attributes of the product. Summary of the invention

[0005] The first object of the present invention is to provide a method for constructing a characteristic spectrum of a pain tincture.

[0006] The method for constructing the characteristic spectrum of the pain tincture provided by the present invention comprises the following steps:

[0007] a. Preparation of reference solution: Take appropriate amount of bergenin, artemisinin A, rutin, cinnamaldehyde, paeonol and aconitine reference substances, add solvent to dissolve, and obtain reference solution;

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

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

[0010] Furthermore, in step a, the preparation method of the reference solution is to accurately weigh bergenin, artemisinin A, rutin, cinnamaldehyde, paeonol and aconitine reference substances, and add methanol to obtain a reference solution containing 0.05 mg of bergenin, 0.02 mg of artemisinin A, 0.02 mg of rutin, 1 mg of cinnamaldehyde, 0.08 mg of paeonol and 0.02 mg of aconitine per 1 mL.

[0011] Furthermore, in step b, the preparation method of the test solution is to accurately measure 2 mL of the pain tincture into a 10 mL volumetric flask, dilute it with an ethanol solution with a volume concentration of 80-90% and make up to the scale, shake well, filter, 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, acetonitrile as mobile phase B, and gradient elution is adopted, and the gradient elution program is as follows: 0-25min, 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%; 25-30min, 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%; 30-30.1min, 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%; 30.1-35min, 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°C; the detection wavelength is 190-400nm; the flow rate is 0.3-0.75mL / min; 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 selected in sections: 0-7min, wavelength is 210nm; 7.01-10min, wavelength is 220nm; 10.01-14min, wavelength is 270nm; 14.01-16min, wavelength is 337nm; 16.01-19.5min, wavelength is 230nm; 19.51-35min, wavelength is 210nm.

[0015] The second object of the present invention is to provide a method for detecting the pain tincture.

[0016] The detection method of the pain tincture provided by the present invention comprises the following steps: accurately aspirating a test solution of the pain tincture, determining it by ultra-high performance liquid chromatography, and comparing the obtained ultra-high performance liquid chromatogram with the characteristic spectrum of the pain tincture obtained by the characteristic spectrum construction method;

[0017] The ultra-high performance liquid chromatography method adopts a mobile phase with 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B, and adopts gradient elution, and the gradient elution program is as follows: from 0 to 25 minutes, 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%; from 25 to 30 minutes, 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%; from 30 to 30.1 minutes, 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%; from 30.1 to 35 minutes, 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 to 40°C; the detection wavelength is 190 to 400nm; the flow rate is 0.3 to 0.75mL / min; and the detector is a diode array detector.

[0018] Preferably, the chromatographic column is 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 selected in sections: 0-7min, wavelength is 210nm; 7.01-10min, wavelength is 220nm; 10.01-14min, wavelength is 270nm; 14.01-16min, wavelength is 337nm; 16.01-19.5min, wavelength is 230nm; 19.51-35min, wavelength is 210nm.

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

[0022] The beneficial effects of the present invention are:

[0023] The present invention adopts ultra-high performance liquid chromatography, combined with appropriate detection conditions, to construct a UPLC characteristic spectrum of the pain tincture, and determines bergenin, artemisinin, rutin, cinnamaldehyde, paeonol and aconitine, and 13 common peaks belonging to hibiscus leaves, Cynanchum chinense, Zanthoxylum bungeana, cinnamon root, artemisia annua, camphor, and cinnamon oil. The characteristic spectrum constructed by the method of the present invention can fully reflect the characteristic peak information of the pain tincture, and the method is stable, with high precision and good reproducibility, and can quickly and effectively comprehensively evaluate the quality of the pain tincture, providing a practical reference method for the quality control evaluation of the pain tincture. The present invention also provides a detection method for the pain tincture, by comparing the spectrum of the pain tincture product to be tested with the control characteristic spectrum, the characteristic effective components of the pain tincture are quality tested, so that the quality test of the pain tincture is more comprehensive, and the effectiveness and controllability of its quality are guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The characteristic spectrum of the pain tincture established for the present invention (presenting 13 characteristic peaks, peak 2 is bergenin (Cinnabaris root), peak 3 is artemisinin (Artemisia annua), peak 5 is rutin (Hibiscus leaf), peak 7 is cinnamaldehyde (Cinnamon oil), peak 8 is paeonol (Cynanchum chinense), peak 11 is aconitine (Artemisia annua), peak 1 belongs to Hibiscus leaf, peak 4 belongs to Zanthoxylum bungeana, peak 6, peak 9, peak 10, peak 13 belong to cinnamon oil, and peak 12 belongs to camphor).

[0025] Figure 2 This is the identification spectrum of the chromatographic peak of the pain tincture test sample using the reference substance.

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

[0027] Figure 4 This is the aqueous phase investigation spectrum of the mobile phase in Example 2.

[0028] Figure 5 This is the investigation spectrum of the chromatographic column in Example 2.

[0029] Figure 6 This is the investigation spectrum of column temperature in Example 2.

[0030] Figure 7 This is the investigation spectrum of the detection wavelength in Example 2.

[0031] Figure 8 It is the characteristic peak attribution spectrum of the pain tincture in Example 2.

[0032] Fig. 9 This is the confirmed spectrum of the common peaks of the characteristic spectrum in Example 2.

[0033] Fig.10 This is the precision investigation spectrum in Example 2.

[0034] Fig.11 This is a repeatability test spectrum in Example 2.

[0035] Fig.12 This is the stability investigation spectrum in Example 2. DETAILED DESCRIPTION

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

[0037] Example 1

[0038] 1. Instruments and test drugs

[0039] 1.1 Instrument

[0040] Agilent 1290 Agilent Technologies (China) Co., Ltd., 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 premium grade; water was distilled water from Watsons; and anhydrous ethanol was of analytical grade.

[0043] 1.3 Reference substances

[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 purchased from the China Food and Drug Administration. Aconitine (A2311005, purity 98.15%, 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°C; flow rate 0.75mL / min, detector is diode array detector; segmented detection wavelength is shown in Table 2; injection volume 1uL, theoretical plate number calculated according to cinnamaldehyde peak should not be less than 130000.

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

[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 wavelength selection in sections

[0051] Time min Wavelength nm Contains 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 test solution:

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

[0054] 2.3 Preparation of reference solution

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

[0056] 2.4 Determination method

[0057] Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, measure, record the chromatogram, and obtain the characteristic spectrum of the pain tincture. 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 phase (methanol, acetonitrile) and aqueous phase (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:

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

[0063] 25-30 min, the volume fraction of 0.1% phosphoric acid solution decreased from 50% to 30%, and the volume fraction of acetonitrile increased from 50% to 70%;

[0064] 30-30.1 min, the volume fraction of 0.1% phosphoric acid solution increased from 30% to 97%, and the volume fraction of acetonitrile decreased from 70% to 3%;

[0065] 30.1~35min, the volume fraction of 0.1% phosphoric acid solution is maintained at 97%, and the volume fraction of acetonitrile is maintained at 3%.

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

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

[0068] 25-30 min, the volume fraction of 0.1% phosphoric acid solution decreased from 20% to 10%, and the volume fraction of methanol increased from 80% to 90%;

[0069] 30-30.1min, the volume fraction of 0.1% phosphoric acid solution increased from 10% to 95%, and the volume fraction of methanol decreased from 90% to 5%;

[0070] 30.1~35min, maintain the volume fraction of 0.1% phosphoric acid solution at 95% and the volume fraction of methanol at 5%.

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

[0072] From 0 to 25 min, the volume fraction of water decreased from 97% to 50%, and the volume fraction of acetonitrile increased from 3% to 50%;

[0073] 25-30 min, the volume fraction of water decreased from 50% to 30%, and the volume fraction of acetonitrile increased from 50% to 70%;

[0074] 30-30.1min, the volume fraction of water increased from 30% to 97%, and the volume fraction of acetonitrile decreased from 70% to 3%;

[0075] 30.1~35min, keep the volume fraction of water at 97% and the volume fraction of acetonitrile at 3%;

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

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

[0078] 25-30 min, the volume fraction of 0.1% formic acid solution decreased from 50% to 30%, and the volume fraction of acetonitrile increased from 50% to 70%;

[0079] 30-30.1 min, the volume fraction of 0.1% formic acid solution increased from 30% to 97%, and the volume fraction of acetonitrile decreased from 70% to 3%;

[0080] 30.1~35min, the volume fraction of 0.1% formic acid solution is maintained at 97%, and the volume fraction of acetonitrile is maintained at 3%.

[0081] Results Figure 3 , Figure 4 Finally, acetonitrile was selected as the organic phase and 0.1% phosphoric acid solution was selected as the aqueous phase for gradient elution, and the characteristic peaks were better separated and the baseline was flat.

[0082] 2.2 Selection of chromatographic columns

[0083] The inventors investigated ultra-high performance liquid chromatography columns of 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) A chromatographic column Agilent 120Aq-C18 was used, and the gradient elution program in Table 1 of Example 1 was followed, with 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 nm: 210→220→270→337→230→210.

[0087] (2) A chromatographic column thermo GOLD (2.1 mm × 100 mm, 1.9 μm) was used according to the gradient elution program in Table 1 of Example 1, with a flow rate of 0.30 mL / min. The segmented 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 nm: 210→220→270→337→230→210.

[0090] (3) A chromatographic column Waters HSS T3 (2.1 mm × 100 mm, 1.8 μm) was used according to the gradient elution program in Table 1 of Example 1, with 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 nm: 210→220→270→337→230→210.

[0093] The results show that the Agilent 120Aq-C18 (3.0mm×150mm, 2.7μm) column has the best separation effect on the sample and can achieve baseline separation of all characteristic peaks in the sample. Figure 5 .

[0094] 2.3 Selection of column temperature

[0095] The inventors investigated 30℃, 35℃, and 40℃. The results showed that when the column temperature was 35℃ and 40℃, all characteristic peaks could achieve baseline separation and the separation effect was good. Therefore, it is more appropriate to select a column temperature between 35℃ and 40℃. Figure 6 .

[0096] 2.4 Selection of detection wavelength

[0097] The detection conditions of Example 1 of the present invention are adopted, and the sample is scanned at full wavelength in the range of 190nm to 400nm using a PDA detector. According to the principle of maximizing effective information, the wavelength that can best reflect the sample component information is selected.

[0098] The results show that the chromatographic peaks contained in the sample cannot have corresponding absorption at a fixed wavelength, and cinnamaldehyde has large absorption at multiple wavelengths, with extremely high response values, resulting in low response values ​​of other chromatographic peaks in the same chromatogram. Therefore, according to the absorption value of each chromatographic peak, an appropriate wavelength is selected to achieve as many chromatographic peaks as possible, with greater absorption, richer information, stronger characteristics, and more beautiful overall spectrum, so the following segmented wavelengths are selected:

[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. Figure 7 .

[0101] 2.5 Attribution of characteristic peaks

[0102] Adopting the detection conditions of Example 1 of the present invention, sample the reference solutions of bergenin, artemisinin A, rutin, cinnamaldehyde, paeonol and aconitine and the test solution of the pain tincture for detection, record the chromatogram, compare the above-mentioned reference substances with the test sample spectrum, and identify the chemical components in the sample according to the retention time. Result: Peak 2 is bergenin (Cinnabarin root), Peak 3 is artemisinin A (Artemisia annua), Peak 5 is rutin (Hibiscus leaf), Peak 7 is cinnamaldehyde (Cinnamon oil), Peak 8 is paeonol (Cynanchum paniculatum), Peak 11 is aconitine (Artemisia annua), Peak 1 belongs to Hibiscus leaf, Peak 4 belongs to Zanthoxylum bungeana, Peak 6, Peak 9, Peak 10, Peak 13 belong to cinnamon oil, and Peak 12 belongs to camphor. See Figure 8 .

[0103] 2.6 Determination of common peaks in characteristic spectra

[0104] Using the detection conditions of Example 1 of the present invention, 13 batches of pain tincture (batch numbers: 231101, 231103, 231106, 231201, 231205, 240302, 240304, 240501, 240502, 240504, 240603, 240702, 240705) were taken and the retention time of each characteristic peak was determined according to the method of the present invention. There should be 13 characteristic peaks in the test solution, and the peak No. 7 corresponding to the cinnamaldehyde reference substance is the S peak. The relative retention time of the remaining characteristic peaks and the S peak is calculated. The RSD of the retention time is between 0.01% and 0.11%, and the RSD of the relative retention time is between 0.00% and 0.12%, indicating that the batch consistency of the 13 batches of pain tincture is good. The results are shown in Tables 3 to 6. Fig. 9 .

[0105] Table 3 Retention time of peak 1 to peak 8 of 13 batches of samples (min)

[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 time of peak 9 to peak 13 of 13 batches of samples (min)

[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 time of peak 1 to peak 8 of 13 batches of samples (min)

[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 time of peak 9 to peak 13 of 13 batches of samples (min)

[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 investigation

[0115] Take 6 mixed reference solutions of bergenin, artemisinin A, rutin, cinnamaldehyde, paeonol and aconitine, and inject 5 injections continuously according to the detection conditions of Example 1 of the present invention, each injection is 1 μL. Among them, peak 2, peak 3, peak 5, peak 7, peak 8, peak 11 correspond to the retention time of reference substances bergenin, artemisinin A, rutin, cinnamaldehyde, paeonol and aconitine, respectively. Peak No. 7 corresponding to the cinnamaldehyde reference substance is S peak. Record the retention time and peak area, calculate the RSD value of retention time and peak area, the retention time RSD is between 0.00% and 0.02%, and the peak area RSD is between 0.39% and 0.69%, indicating good precision. The results are shown in Tables 7 and 8. Fig.10 .

[0116] Table 7 Precision - 6 mixed control retention times (min)

[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 - 6 mixed control peak areas

[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 study

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

[0122] Table 9 Repeatability Study - Retention Time of Peak 1 to Peak 8 (min)

[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 Repeatability Study - Retention Time of Peak 9 to Peak 13 (min)

[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 study - relative retention time of peak 1 to peak 8 (min)

[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 Repeatability study - relative retention time of peak 9 to peak 13 (min)

[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 test solution

[0131] According to the detection conditions of Example 1 of the present invention, the pain tincture (batch number: 240501) was tested, and the test solution was prepared according to the test solution preparation method of the present invention. The retention time and peak area were measured at 0h, 5h, 10h, 15h, 20h, 25h, 30h, and 35h, and the RSD values ​​of the retention time and peak area were calculated. The retention time RSD was between 0.02% and 0.15%, and the peak area RSD value 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. Fig.12 .

[0132] Table 13 Stability Study - Retention Time of Peak 1 to Peak 8 (min)

[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 Time of Peak 9 to Peak 13 (min)

[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 to 8

[0137]

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

[0139]

[0140] Example 4

[0141] Take 13 batches of pain tincture (batch numbers: 231101, 231103, 231106, 231201, 231205, 240302, 240304, 240501, 240502, 240504, 240603, 240702, 240705) and prepare the sample solution according to "2.2 Preparation of test solution" in Example 1. Accurately pipette the sample solution and inject it into an ultra-high performance liquid chromatograph for determination. The chromatographic conditions are the same as those in "2.1 Chromatographic conditions" in Example 1. The test results are shown in FIG. Fig. 9 and Tables 3 to 6.

[0142] In summary, the characteristic spectrum constructed by the present invention can fully reflect the characteristic peak information of the pain tincture, and the method is stable, with high precision and good reproducibility, and can quickly and effectively comprehensively evaluate the quality of the pain tincture. The detection method of the pain tincture of the present invention compares the spectrum of the pain tincture product to be tested with the control characteristic spectrum, and performs quality detection on the characteristic effective components of the pain tincture, so that the quality detection of the pain tincture is more comprehensive, and the effectiveness and controllability of its quality are guaranteed.

Claims

1. A method for constructing a characteristic spectrum of a pain tincture, characterized in that: The steps include: a. Preparation of reference solution: Take appropriate amount of bergenin, artemisinin A, rutin, cinnamaldehyde, paeonol and aconitine reference substances, add solvent to dissolve, and obtain reference solution; b. Preparation of test solution: Take pain tincture and add ethanol solution to prepare; c. Detection: Inject the reference solution and the test solution into an ultra-high performance liquid chromatograph for detection. Using the characteristic spectrum of the reference solution as a reference spectrum, select common peaks from the characteristic spectrum of the test solution to construct the characteristic spectrum of the pain tincture.

2. The method for constructing a characteristic spectrum of the pain tincture according to claim 1, characterized in that: In step a, the preparation method of the reference solution is to accurately weigh the reference substances of bergenin, artemisinin A, rutin, cinnamaldehyde, paeonol and aconitine, and add methanol to obtain a reference solution containing 0.05 mg of bergenin, 0.02 mg of artemisinin A, 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 a characteristic spectrum of the pain tincture according to claim 1, characterized in that: In step b, the preparation method of the test solution is to accurately measure 2 mL of the pain tincture into a 10 mL volumetric flask, dilute it with an ethanol solution with a volume concentration of 80-90% and make up to the scale, shake well, filter, and take the filtrate to obtain the test solution.

4. The method for constructing a characteristic spectrum of the pain tincture according to claim 1, characterized in that: In step c, the detection conditions of the ultra-high performance liquid chromatography are: The mobile phase used 0.1% phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B. Gradient elution was used, and the gradient elution program was as follows: from 0 to 25 minutes, 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%; from 25 to 30 minutes, 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%; From 30 to 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%; 30.1~35min, maintain the volume fraction of mobile phase A at 97%, and the volume fraction of mobile phase B at 3%; column temperature is 30~40℃; detection wavelength is 190~400nm; flow rate is 0.3~0.75mL / min; detector is a diode array detector.

5. The method for constructing a characteristic spectrum of the pain tincture according to claim 4, characterized in that: The detection wavelength is selected in sections: 0-7min, wavelength is 210nm; 7.01-10min, wavelength is 220nm; 10.01-14min, wavelength is 270nm; 14.01-16min, wavelength is 337nm; 16.01-19.5min, wavelength is 230nm; 19.51-35min, wavelength is 210nm.

6. The method for constructing a characteristic spectrum of the pain 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 of pain tincture is characterized by: The method comprises the following steps: accurately aspirating a test solution of the pain tincture, determining the solution by ultra-high performance liquid chromatography, and comparing the obtained ultra-high performance liquid chromatogram with the characteristic spectrum of the pain tincture obtained by the characteristic spectrum construction method according to any one of claims 1 to 6; The mobile phase used in the ultra-high performance liquid chromatography method is 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution. The gradient elution program is as follows: from 0 to 25 minutes, 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%; from 25 to 30 minutes, 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%; From 30 to 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%; 30.1~35min, maintain the volume fraction of mobile phase A at 97%, and the volume fraction of mobile phase B at 3%; column temperature is 30~40℃; detection wavelength is 190~400nm; flow rate is 0.3~0.75mL / min; detector is a diode array detector.

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

9. The method for constructing a characteristic spectrum of the pain tincture according to claim 7, characterized in that: In step c, the column temperature is 35-40° C. and the flow rate is 0.75 mL / min.

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

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