Method for constructing characteristic chromatogram of fumai tablet and application of method in quality control method

Abstract

The application discloses a characteristic spectrum construction method of Fumai tablets and application of the method in a quality control method. The application provides the characteristic spectrum construction method of the Fumai tablets, which comprises the following steps: preparing a Fumai tablet test sample solution: weighing Fumai tablet extract, adding a methanol-water solution, ultrasonic extraction, centrifugation, taking supernatant, filtration, and obtaining the test sample solution; detecting the test sample solution by using an ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer, and collecting a total ion current chromatogram; performing peak extraction, peak alignment and peak correction on the total ion current chromatogram, and establishing a characteristic spectrum of the Fumai tablets. The characteristic spectrum construction method effectively solves the blank problem of the current quality evaluation method of the Fumai tablets, and as one of core technologies for quality evaluation of the Fumai tablets, realizes precise control of the quality of the Fumai tablets from four dimensions of characteristics, identification, inspection and qualitative analysis, and provides data support for establishment and application of the quality standard of the Fumai tablets.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically involving the construction method of characteristic chromatograms of Fusong tablets (a traditional Chinese medicine compound preparation for the treatment of systemic sclerosis) and its application in quality control methods. Background Technology

[0002] Fusong tablets are a compound preparation composed of six traditional Chinese medicines: Euonymus alatus, Astragalus membranaceus, Angelica sinensis, Pueraria lobata, Ligusticum chuanxiong, and Carthamus tinctorius. It has been used clinically for decades and has significant value. However, due to the large variety of medicinal materials and complex chemical composition of Fusong tablets, including flavonoids, organic acids, saponins, and phthalides, the identification of its effective chemical components has remained a gap.

[0003] The existing quality evaluation method for Fusong tablets is based on the thin-layer chromatography method specified in the pharmacopoeia for Astragalus membranaceus and Puerarin. However, the other four Chinese medicinal materials are not subject to quality control, making it difficult to fully reflect their chemical composition, effectively guarantee the uniformity and stability of product quality, and hindering the elucidation of their pharmacodynamic material basis and the assurance of clinical efficacy.

[0004] Characteristic mapping technology, as one of the core technologies for the quality evaluation of traditional Chinese medicine (TCM), can comprehensively and systematically reflect the chemical composition characteristics of TCM, providing a scientific basis for TCM quality evaluation. Currently, there are no reports on methods for establishing characteristic maps of cyproconazole tablets. Therefore, establishing a stable, reliable, and comprehensive method for establishing characteristic maps of cyproconazole tablets and applying it to quality evaluation and efficacy research is of great significance for ensuring the quality and clinical efficacy of cyproconazole tablets. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing the characteristic spectrum of lysine tablets and its application in quality evaluation and efficacy research.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for constructing a characteristic map of pine nut tablets, comprising the following steps: a) Preparation of the test solution of compound pine tablets: Weigh the compound pine tablet extract, add methanol-water solution, extract by ultrasonication, centrifuge, take the supernatant, filter, and obtain the test solution; b) The test solution was analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry, and a total ion chromatogram was collected; c) Perform peak extraction, peak alignment, and peak correction on the total ion chromatogram to establish a characteristic spectrum of Fusong tablets; The chromatographic conditions for the ultra-high performance liquid chromatography include: The chromatographic column was a CORTECS T3 column; Mobile phase A is an aqueous solution containing formic acid, and mobile phase B is an acetonitrile solution containing formic acid.

[0007] In one specific embodiment, in step a), the extract of pine oxytocin tablets is weighed, placed in a centrifuge tube, 50-80% methanol aqueous solution is added, ultrasonic treatment is performed for 15-30 min, centrifugation is performed, the supernatant is collected, filtered, and a test solution of 20 mg / mL is obtained.

[0008] The chromatographic conditions for the ultra-high performance liquid chromatography also include: column specifications: 100 mm × 2.1 mm, 1.6 μm; mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; column temperature: 35℃; sample chamber temperature: 10℃; flow rate: 0.3 mL / min; injection volume: 2 μL.

[0009] In one specific embodiment, the gradient elution program in the chromatographic conditions of ultra-high performance liquid chromatography is as follows: at 0.0 min, A is 99.0% and B is 1.0%; at 5.0 min, A is 90.0% and B is 10.0%; at 15.0 min, A is 86.0% and B is 14.0%; at 18.0 min, A is 86.0% and B is 14.0%; at 22.0 min, A is 84.0% and B is 16.0%; at 24.0 min, A is 70.0% and B is 30.0%; at 30.0 min, A is 65.0% and B is 35.0%; at 34.0 min, A is 65.0% and B is 35.0%; at 35.0 min, A is 45.0% and B is 55.0%; at 50.0 min, A is 30.0% and B is 1 ... At 55.0 min, the percentage of A was 70.0%; at 55.0 min, the percentage of B was 10.0% and the percentage of A was 90.0%.

[0010] The mass spectrometry conditions of the ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometer include: an electrospray ionization source; scanning in both positive and negative ion modes; capillary voltage of 3.0 kV for positive ion mode and 2.3 kV for negative ion mode; cone voltage of 40 V; extraction cone voltage of 3 V; ion source temperature of 100 °C; desolvation gas temperature of 400 °C for positive ion mode and 350 °C for negative ion mode; desolvation gas flow rate of 600 L / h; collision gas flow rate of 0.5 mL / min; scan time of 0.5 s; scan interval of 0.02 s; mass-to-charge ratio scan range of 50-1200 m / z; and locked mass numbers of [M+H]+ 556.2771 and [MH]. 554.2615.

[0011] The characteristic spectrum contains 22 characteristic peaks, with retention times as follows: Peak 1 (sucrose) 0.87 min, Peak 5 (citric acid) 1.05 min, Peak 6 (isocitric acid) 1.41 min, Peak 11 (adenine) 2.28 min, Peak 17 (neochlorogenic acid) 4.7 min, Peak 18 (caffeic acid) 5.06 min, Peak 19 (tanshinone F) 5.54 min. Peak 25: Chlorogenic acid 6.24 min; Peak 27: Cryptochlorogenic acid 6.65 min; Peak 38: Puerarin 8.28 min; Peak 45: Ferulic acid 10.05 min; Peak 50: Daidzidin 10.80 min; Peak 63: Verbena isoflavone glucoside 14.19 min; Peak 76: 3,4-dicaffeoylquinic acid 17.23 min; Peak 78: Ligusticum lactone F 18.03 min; Peak 89: 3,5-dicaffeoylquinic acid 23.45 min; Peak 94: Ammonium glutamicum 24.2 min; Peak 96: Daidzidin 24.46 min; Peak 105: Naringenin 26.1 min; Peak 115: Ammonium glutamicum 28.65 min; Peak 141: E-Ligustilide 37.12 min; Peak 142: n-Butylphthalide 37.22 min.

[0012] The characteristic spectrum also contains 115 characteristic peaks, representing a total of 137 compounds.

[0013] Table 1 shows 137 compounds corresponding to the characteristic chromatograms.

[0014] * was verified by a reference standard.

[0015] Secondly, the present invention provides a quality control method for pine nut tablets, comprising the following steps: (1) Provide a reference feature spectrum for lysine tablets; the reference feature spectrum is generated by testing multiple batches of qualified lysine tablet samples using the feature spectrum construction method described in any one of claims 1-5; (2) Using the feature map construction method according to any one of claims 1-5, a feature map of the sample of the pine nut tablets to be tested is constructed; (3) Compare the characteristic spectrum of the sample to be tested obtained in step (2) with the control characteristic spectrum obtained in step (1), and determine the quality of the sample to be tested based on the retention time and / or peak area matching of the common peak.

[0016] In one specific embodiment, in step (3), the feature spectrum of the sample to be tested obtained in step (2) can be compared with the control feature spectrum to evaluate the similarity and calculate the similarity value; then the quality consistency of the sample to be tested is judged according to the similarity value: if the similarity is not less than 0.90, it is judged as qualified.

[0017] The comparison includes matching the characteristic spectrum of the test compound tablet with the characteristic spectrum of one or more control compounds by retention time and / or mass spectrometry information.

[0018] The control compounds include one or more of the following: sucrose, citric acid, isocitrate, adenine, neochlorogenic acid, caffeic acid, salvianolic acid F, chlorogenic acid, cryptochlorogenic acid, puerarin, ferulic acid, daidzein, 3,4-dicaffeoylquinic acid, ligustilide F, 3,5-dicaffeoylquinic acid, daidzein, daidzein, naringenin, daidzein, E-ligustilide, n-butylphthalide, and verrucoside.

[0019] The quality assessment includes one or more of the following: finished product appearance inspection, finished product identification inspection, finished product n-butanol extract content inspection, and semi-finished product qualitative inspection.

[0020] In one specific embodiment, the qualitative inspection of the semi-finished product includes: establishing a characteristic spectrum of the semi-finished product solution to be tested using the characteristic spectrum construction method described in this invention, and comparing it with the control characteristic spectrum to determine the consistency between the semi-finished product solution to be tested and the control standard sample.

[0021] The finished product characteristics inspection includes visual inspection and smell and taste (previously, it was a yellow sugar coating).

[0022] The finished product identification and inspection: (1) Take 15 tablets of this product, remove the sugar coating, grind finely, add 40 ml of chloroform, heat under reflux for 1 hour, filter, discard the filtrate, evaporate the residue to dryness, add 40 ml of methanol, heat under reflux for 1 hour, filter, and evaporate the filtrate to dryness. Add 30 ml of water to the residue and heat gently to dissolve, extract twice with water-saturated n-butanol, 25 ml each time, combine the n-butanol solutions, wash twice with 1% sodium hydroxide solution, 20 ml each time, discard the alkali solution, wash twice with water-saturated n-butanol, 15 ml each time, discard the aqueous solution, evaporate the n-butanol solution to dryness, add 1 ml of methanol to the residue to dissolve, and use as the test solution; separately take astragaloside A reference standard, add methanol to prepare a solution containing 1 mg per 1 ml, and use as the reference solution; Thin-layer chromatography (Chinese Pharmacopoeia, current edition) was used. 5 μl of each of the two solutions were spotted separately onto the same silica gel G thin-layer plate. The lower layer of a chloroform-methanol-water (13:7:2) solution was used as the developing solvent. After development, the plate was removed, dried, sprayed with 10% sulfuric acid in ethanol, and heated at 105°C until the spots were clearly visible. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference sample.

[0023] (2) Take 10 tablets of this product, remove the sugar coating, grind into a fine powder, add 30 ml of methanol, shake well, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, and use it as the test solution; take puerarin reference standard, add methanol to prepare a solution containing 1 mg per 1 ml, and use it as the reference solution. Thin-layer chromatography (Chinese Pharmacopoeia, current edition) was used. 2 μl of the test solution and 5 μl of the reference solution were spotted separately onto the same silica gel G thin-layer plate. The plate was developed using chloroform-methanol-water (28:10:1) as the developing solvent. The plate was then removed, dried, and examined under ultraviolet light (365 nm). Fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the test solution and the reference solution.

[0024] The content test of the n-butanol extract of the finished product is as follows: Take an appropriate amount of this product (25 tablets), remove the sugar coating, grind it into a fine powder, take 3g, accurately weigh it, place it in a stoppered conical flask, add 50ml of methanol, heat it in a water bath under reflux for 1 hour, remove it, cool it, filter it, wash the residue and filter with 20ml of methanol several times, combine the filtrate and washings, evaporate to dryness, add 20ml of water to dissolve the residue, extract it three times with water-saturated n-butanol, 25ml each time, combine the n-butanol solutions, place them in an evaporating dish that has been dried to constant weight, evaporate to dryness, dry at 105℃ for 3 hours, transfer it to a desiccator, cool it for 30 minutes, quickly and accurately weigh it, and calculate the result. The content of the n-butanol extract in this product should not be less than 7.5%.

[0025] The compounds identified in the characteristic chromatograms are used to screen for potential active ingredients in lysine tablets for the treatment of systemic sclerosis.

[0026] The potential active ingredients include butyl paraben, naringenin, and caffeic acid, with Degree values ​​of 16, 11, and 9, respectively.

[0027] Furthermore, the specific steps of the network pharmacology analysis include: using compounds identified by characteristic spectra as candidate components, obtaining Isomeric SMILES and 2D structures through PubChem, and predicting targets using Swiss TargetPrediction; using "Systemic sclerosis" as a keyword, obtaining disease targets through GeneCards and OMIM databases; taking the intersection of drug targets and disease targets, constructing a PPI network using STRING 12.0, and screening core targets with Degree values ​​greater than the quartiles using Cytoscape 3.9.1; uploading the core targets to the Metascape platform for KEGG pathway enrichment analysis, obtaining 259 signaling pathways such as the MAPK signaling pathway and the Neurotrophin signaling pathway.

[0028] Compared with the prior art, the beneficial effects achieved by the present invention are: The characteristic spectrum construction method proposed in this invention is the first to systematically identify the chemical components of cyproconazole tablets, qualitatively identifying 137 chemical components in a single step, fully covering all pharmacodynamic groups such as flavonoids, saponins, organic acids, and phthalides. This characteristic spectrum construction method for cyproconazole tablets effectively addresses the gap in current quality evaluation methods. As one of the core technologies for cyproconazole tablet quality evaluation, it comprehensively and precisely controls the quality of cyproconazole tablets from four dimensions: appearance, identification, inspection, and qualitative analysis, providing data support for the establishment and application of quality standards for cyproconazole tablets. Attached Figure Description

[0029] Figure 1 The chromatograms are of the lysine tablets; where (a) represents the positive ion mode base peak chromatogram and (b) represents the negative ion mode base peak chromatogram.

[0030] Figure 2 Mass spectrometry fragment ion diagram (negative ion) of kaempferol-3-O-rhamnosylglucose-7-O-rhamnoside.

[0031] Figure 3 This is a mass spectrometry fragment ion diagram (negative ion) of chlorogenic acid.

[0032] Figure 4 Mass spectrometry fragment ion diagram (positive and negative ions) of 3'-hydroxypuerarin.

[0033] Figure 5 Mass spectrometry fragment ion diagram (positive and negative ions) of apigenin-5-O-neohesperidin.

[0034] Figure 6 This is a mass spectrometry fragment ion diagram (positive and negative ions) of puerarin.

[0035] Figure 7 This is a mass spectrometry fragment ion diagram (negative ions) of kaempferol.

[0036] Figure 8 Mass spectrometry fragment ion diagram (positive and negative ions) of 3'-methoxypuerarin.

[0037] Figure 9 Mass spectrometry fragment ion diagrams (positive and negative ions) of puerarin apigenin and puerarin-6''-O-xyloside.

[0038] Figure 10 This is a mass spectrometry fragment ion diagram (positive and negative ions) of daidzin.

[0039] Figure 11 Mass spectrometry fragment ion diagram (positive and negative ions) of 6''-O-acetylasdin.

[0040] Figure 12 This is a mass spectrometry fragment ion diagram (positive and negative ions) of genistein.

[0041] Figure 13 Mass spectrometry fragment ion diagram (negative ion) of 3,4-dicaffeoylquinic acid.

[0042] Figure 14 This is a mass spectrometry fragment ion diagram (positive ion) of ligustilide F.

[0043] Figure 15 This is a mass spectrometry fragment ion diagram (positive and negative ions) of gentianin.

[0044] Figure 16 This is a mass spectrometry fragment ion diagram (positive and negative ions) of puerarin C.

[0045] Figure 17 This is a mass spectrometry fragment ion diagram (positive and negative ions) of daidzein C.

[0046] Figure 18 This is a mass spectrometry fragment ion diagram (positive and negative ions) of verbascoside isoflavone.

[0047] Figure 19 Mass spectrometry fragment ion diagram (positive and negative ions) of 3'-methoxydaidzein.

[0048] Figure 20 This is a mass spectrometry fragment ion diagram (positive and negative ions) of gentianin.

[0049] Figure 21 This is a mass spectrometry fragment ion diagram (positive and negative ions) of astragaloside III.

[0050] Figure 22This is a mass spectrometry fragment ion diagram (positive and negative ions) of cycloastragaloside glucoside.

[0051] Figure 23 This is a mass spectrometry fragment ion diagram (positive ion) of soybean saponin Bd.

[0052] Figure 24 This is a mass spectrometry fragment ion diagram (positive and negative ions) of astragaloside II.

[0053] Figure 25 This is a mass spectrometry fragment ion diagram (positive and negative ions) of soybean saponin I.

[0054] Figure 26 This is a mass spectrometry fragment ion diagram (positive and negative ions) of soybean saponin III.

[0055] Figure 27 This is a mass spectrometry fragment ion diagram (positive ion) of soybean saponin II.

[0056] Figure 28 This is a mass spectrometry fragment ion diagram (positive ion) of soybean saponin Be.

[0057] Figure 29 This is a mass spectrometry fragment ion diagram (negative ion) of soybean saponin glucuronide.

[0058] Figure 30 Mass spectrometry fragment ion diagram (positive and negative ions) of soybean saponin glucuronide. Detailed Implementation

[0059] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0062] Example 1 1. Materials and Instruments 1.1 Experimental materials: The experimental sample of pine oxytocin tablet extract (batch number: 20230825) was provided by Peking Union Medical College Hospital.

[0063] 1.2 Instruments: SYNAPT XS HDMS ultra-high performance liquid chromatography-time-of-flight high-resolution mass spectrometry system (Waters Corporation, Milford, MA, USA), data processing system is MarkerLynx 4.2 workstation (Waters, Manchester, UK), AB135-S electronic analytical balance (Mettler-Toledo), KQ-118B ultrasonic oscillator (Kunshan Ultrasonic Instrument Co., Ltd.).

[0064] 1.3 Reagents: Chromatographic acetonitrile and methanol were purchased from JT Baker (Phillipsburg, NJ, USA); chromatographic formic acid, phosphoric acid, and leucine enkephalin were purchased from Sigma Aldrich (MO, USA); experimental ultrapure water (18.2 MΩ) was prepared using a Milli-Q water purification system (Millipore, France); and all other reagents used were of analytical grade.

[0065] 2. Experimental Methods and Results 2.1 Preparation of test solution: Weigh 40 mg of pine nitrate extract and place it in a 2 mL centrifuge tube. Add 2 mL of 50% methanol-water mixture and sonicate for 30 min. After centrifugation, take the supernatant and filter it through a 0.22 μm organic phase microporous membrane to obtain the test solution with a concentration of approximately 20 mg / mL.

[0066] 2.2 Preparation of reference solutions: Accurately weigh appropriate amounts of sucrose, citric acid, isocitrate, adenine, neochlorogenic acid, caffeic acid, salvianolic acid F, chlorogenic acid, cryptochlorogenic acid, puerarin, ferulic acid, daidzein, 3,4-dicaffeoylquinic acid, ligustrol F, 3,5-dicaffeoylquinic acid, daidzein, daidzein, naringenin, daidzein, E-ligustilide, n-butylphthalide, and verbascoside reference standards (22 in total), dissolve in methanol to prepare a reference stock solution with a mass concentration of 1 mg / mL, and filter through a 0.22 μm microporous membrane to obtain the solution. 2.3 Ultra-high performance liquid chromatography (UPLC) analysis conditions: A Waters ACQUITY UPLC system with a PDA detector was used; the column was a CORTECS T3 column (100 mm × 2.1 mm, id, 1.6 μm); mobile phase A was water (containing 0.1% formic acid), and mobile phase B was acetonitrile (containing 0.1% formic acid); column temperature was 35℃; sample chamber temperature was 10℃; flow rate was 0.3 mL / min; injection volume was 2 μL (positive / negative ion mode); The gradient elution program was as follows: at 0.0 min, A was 99.0% and B was 1.0%; at 5.0 min, A was 90.0% and B was 10.0%; at 15.0 min, A was 86.0% and B was 14.0%; at 18.0 min, A was 86.0% and B was 14.0%; at 22.0 min, A was 84.0% and B was 16.0%; at 24.0 min, A was 70.0% and B was 30.0%; at 30.0 min, A was 65.0% and B was 35.0%; at 34.0 min, A was 65.0% and B was 35.0%; at 35.0 min, A was 45.0% and B was 55.0%; at 50.0 min, A was 30.0% and B was 70.0%; at 55.0 min, A... A is 10.0%, B is 90.0%; 2.4 Mass spectrometry (MS) analysis conditions: A Waters SYNAPT XS HDMS system was used, with nitrogen as the nebulizer and cone gas; electrospray ionization in positive and negative ion modes; capillary voltage: 3.0 kV for positive ion mode and 2.3 kV for negative ion mode; cone voltage: 40 V; extraction cone voltage: 3 V; ion source temperature: 100 °C; desolvation gas temperature: 400 °C for positive ion mode and 350 °C for negative ion mode; reverse cone gas flow rate: 50 L / h; desolvation gas flow rate: 600 L / h (positive / negative ion mode); collision gas flow rate: 0.5 mL / min; scan time: 0.5 s; scan interval: 0.02 s; mass-to-charge ratio range: 50–1200 m / z; locked mass numbers: [M+H]+=556.2771, [MH]-=554.2615; 2.5 Characteristic Spectrum Construction: The test solution and reference solution were analyzed using the conditions in steps 2.3 and 2.4, and the data were processed using a MarkerLynx 4.2 workstation. Peak assignments were determined by combining the chromatographic peak retention behavior, precise molecular weight, MS fragmentation information, and reference standard comparison. The characteristic spectrum contained 137 chromatographic peaks, corresponding to 137 compounds. The results are as follows.

[0067] The data acquisition format is continuous, the sensitivity is normal, and the dynamic range is extended.

[0068] The UPLC-Q-TOF / MS chromatograms and the base peak chromatograms in positive and negative ion modes are shown below. Figure 1 a, Figure 1 b.

[0069] 3. Methodological Validation: (1) Precision test: Take the same test solution and repeat the injection 6 times under the above conditions. Using the chlorogenic acid peak as the reference peak, calculate the relative retention time and relative peak area of ​​137 characteristic peaks. The results show that the RSD of the relative retention time is ≤0.5% and the RSD of the relative peak area is ≤5%, indicating that the instrument has good precision. (2) Stability test: The same test solution was sampled and tested at 0, 2, 4, 8, 12 and 24 hours. The chlorogenic acid peak was used as the reference peak. The relative retention time and relative peak area of ​​137 characteristic peaks were calculated. The results showed that the RSD of the relative retention time was ≤0.8% and the RSD of the relative peak area was ≤5%, indicating that the test solution had good stability within 24 hours. (3) Repeatability test: Take 6 portions of the same batch of pine oxytocin tablet extract and prepare 6 portions of test solution in parallel according to the test solution preparation method. Inject the samples for detection. Using the chlorogenic acid peak as the reference peak, calculate the relative retention time and relative peak area of ​​137 characteristic peaks. The results show that the RSD of the relative retention time is ≤0.6% and the RSD of the relative peak area is ≤5%, indicating that the method has good repeatability.

[0070] Example 2: Application of Feature Maps in Quality Assessment This embodiment provides a quality evaluation method for pine nut tablets, including: finished product appearance inspection, finished product identification inspection, finished product n-butanol extract content inspection, and semi-finished product qualitative inspection; wherein: 1. Finished product characteristics inspection: visual inspection and smell and taste (previously yellow sugar coating).

[0071] 2. Finished product identification and inspection: (1) Take 15 tablets of this product, remove the sugar coating, grind into a fine powder, add 40 ml of chloroform, heat under reflux for 1 hour, filter, discard the filtrate, evaporate the residue to dryness, add 40 ml of methanol, heat under reflux for 1 hour, filter, and evaporate the filtrate to dryness. Add 30 ml of water to the residue and heat gently to dissolve. Extract twice with water-saturated n-butanol, 25 ml each time. Combine the n-butanol extracts, wash twice with 1% sodium hydroxide solution, 20 ml each time, discard the alkali, and then wash twice with water-saturated n-butanol, 15 ml each time, discard the aqueous solution. Evaporate the n-butanol extract to dryness, add 1 ml of methanol to dissolve the residue, and use this as the test solution. Separately, take astragaloside A reference standard, add methanol to prepare a solution containing 1 mg per ml, and use this as the reference solution. According to the thin-layer chromatography method (current version of Chinese Pharmacopoeia), take 5 μl of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate. Use the lower layer solution of chloroform-methanol-water (13:7:2) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample. (2) Take 10 tablets of this product, remove the sugar coating, grind finely, add 30 ml of methanol, shake well, sonicate for 30 minutes, filter, evaporate the filtrate to dryness, add 5 ml of methanol to dissolve the residue, and use it as the test solution. Take another puerarin reference standard, add methanol to prepare a solution containing 1 mg per 1 ml, and use it as the reference solution. Perform the thin-layer chromatography test (current edition of the Chinese Pharmacopoeia). Apply 2 μl of the test solution and 5 μl of the reference solution separately to the same silica gel G thin-layer plate. Develop the plate using chloroform-methanol-water (28:10:1) as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (365 nm). The test solution chromatogram should show fluorescent spots of the same color at the corresponding positions as the reference solution chromatogram.

[0072] 3. Test for n-Butanol Extract Content of Finished Product: Take an appropriate amount of this product (25 tablets), remove the sugar coating, grind finely, take 3g, accurately weigh, place in a stoppered conical flask, add 50ml of methanol, heat under reflux in a water bath for 1 hour, remove, cool, filter, wash the residue and filter with 20ml of methanol several times, combine the filtrate and washings, evaporate to dryness, add 20ml of water to dissolve the residue, extract three times with 25ml of water-saturated n-butanol each time, combine the n-butanol solutions, place in an evaporating dish that has been dried to constant weight, evaporate to dryness, dry at 105℃ for 3 hours, transfer to a desiccator, cool for 30 minutes, quickly and accurately weigh, calculate, and obtain the product. The product must contain no less than 7.5% n-butanol extract.

[0073] 4. Qualitative inspection of semi-finished products: Take samples of the new batch of pine oxytocin tablets, prepare test solutions according to the above-mentioned characteristic spectrum construction method, establish characteristic spectra, and compare them with the characteristic spectra of the reference standard; the results show that the retention time drift of 22 characteristic compounds is within ±1 minute and the m / z is within ±0.5, indicating that the quality of the new batch of pine oxytocin tablets is uniform and stable.

[0074] Example 3: Study on the pharmacodynamic material basis This embodiment provides a method for studying the pharmacodynamic material basis using the aforementioned characteristic map construction method, including: using 137 compounds identified by the characteristic map as candidate components, network pharmacology analysis was used to obtain 559 compound targets, 16,546 systemic sclerosis-related targets, and 521 intersection targets, and 43 core targets with a degree value greater than 46 were screened out; KEGG pathway enrichment analysis yielded 259 signaling pathways, mainly involving the MAPK signaling pathway and the Neurotrophin signaling pathway; potential active ingredients such as butylparaben, naringenin, and caffeic acid were screened out, providing a theoretical basis for the clinical application of Resveratrol tablets.

[0075] Example 4: Optimization Condition Experiment I. Liquid Chromatography Conditions 1. Selection of chromatographic column The following column specifications were selected in the experiment: column length: 50 mm / 100 mm / 150 mm; particle size: 1.7 μm / 1.8 μm / 2.5 μm. The results showed that the 50 mm column had insufficient resolution, the 150 mm column had too long an analysis time and too high a back pressure, and the 1.8 μm and 2.5 μm particle size columns had resolutions lower than 1.6 μm. Ultimately, a 100 mm column with a 1.6 μm particle size was selected, as it balances resolution, peak width, run time, and system pressure tolerance, making it the most commonly used and optimal compromise for non-target UPLC-TOF.

[0076] 2. Selection of mobile phase The following mobile phases were selected in the experiment: (1) Acid-free system: pure water / acetonitrile (acid-free); (2) Formic acid system: 0.1% formic acid / acetonitrile solution containing 0.1% formic acid, 0.2% formic acid / acetonitrile solution containing 0.2% formic acid; (3) Ammonium salt system: 5 mM ammonium formate / acetonitrile solution containing 5 mM ammonium formate, 5 mM ammonium acetate / acetonitrile solution containing 5 mM ammonium acetate. The results showed that the positive ion response of the acid-free mobile phase was low and the peak tailing was severe; the noise of the 0.2% formic acid mobile phase increased and the negative ion suppression was too strong; the positive ion peaks of the ammonium salt system mobile phase were numerous and the baseline noise was large. Finally, 0.1% formic acid was selected because it had the strongest positive ion response, the best peak shape, and the cleanest baseline. It is the most reliable and universal system for both positive and negative modes.

[0077] 3. Column temperature selection The following column temperatures were selected in the experiment: 30 ℃, 35 ℃, and 40 ℃. The results showed that the retention was longer and the peak was slightly wider at a column temperature of 30 ℃; some unstable substances degraded and the retention fluctuated greatly at a column temperature of 40 ℃; and the peak shape, stability, and retention time repeatability were the best at a column temperature of 35 ℃.

[0078] 4. Selection of sample chamber temperature The following sample chamber temperatures were selected in the experiment: 4 ℃, 10 ℃, and room temperature. The results showed that condensation was easy to form at 4 ℃ and air bubbles were easy to form in the injection needle. The samples were unstable and had a high risk of degradation at room temperature. The standard metabolomics setting was used at 10 ℃ to balance stability and instrument stability.

[0079] 5. Selection of flow rate The following flow rates were selected in the experiment: 0.2, 0.3, and 0.4 mL / min. The results showed that the peak was too wide and the number of scan points was redundant at 0.2 mL / min; the back pressure was too high and the peak emerged too quickly, resulting in insufficient TOF sampling at 0.4 mL / min; the optimal flow rate was 0.3 mL / min with a 2.1 mm narrow diameter column and TOF, which had the best pressure, peak width, and ionization efficiency.

[0080] 6. Selection of injection volume The following injection volumes were selected in the experiment: 1, 2, and 5 μL. The results showed that 1 μL had low sensitivity; 5 μL had peak overload, strong solvent effect, and obvious ion suppression; 2 μL had no overload, good linearity, and minimal matrix effect.

[0081] II. Mass Spectrometry Conditions (All were selected using ESI-TOF standard tuned gradients, with the optimal compromise ultimately chosen) 1. Ion source: ESI; simultaneous scanning in positive and negative modes. The following ion sources are available for selection in the experiment: (1) APCI, ESI; (2) positive only, negative only, and positive-negative switching. The results showed that APCI had narrow polarity coverage and lost half of the information in single mode; ESI± simultaneous scanning: the standard protocol for non-target metabolomics, with the most comprehensive coverage.

[0082] 2. Selection of capillary voltage The capillary voltages to be selected in the experiment are as follows: (1) Positive: 2.5, 2.8, 3.0, 3.2 kV; (2) Negative: 2.0, 2.3, 2.5 kV; The results showed that too low a voltage resulted in a weak response; too high a voltage resulted in discharge, soaring noise, and instability; while 3.0 / 2.3 kV had the highest response and no discharge, which is the most commonly used and optimal value for Waters TOF.

[0083] 3. Selection of cone hole voltage The following cone voltages were selected for the experiment: 20, 30, 40, 50, and 60 V. Results showed limitations: <30 V resulted in low transmission efficiency and weak response; 40 V resulted in source fragmentation, increased fragmentation, and a decrease in precursor ions; 40 V was a universal value for the full scan, showing the strongest precursor ion concentration with no significant fragmentation.

[0084] 4. Selection of extraction cone orifice The following extraction cone apertures were selected in the experiment: 2, 3, and 4 V. The results showed that only fine-tuning the transport did not affect the pyrolysis; the noise was lowest and the baseline was flattest at 3 V.

[0085] 5. Selection of ion source temperature The following ion source temperatures were selected in the experiment: 80, 100, and 120 ℃. The results showed that the solvent removal was poor at low temperatures; thermal degradation was easy at high temperatures; and the TOF non-target standard fixed value was obtained at 100 ℃.

[0086] 6. Selection of desolvation gas temperature The following desolvation gas temperatures are available for selection in the experiment: (1) Positive: 350, 400, 450 ℃; (2) Negative: 300, 350, 400 ℃; The results showed that negative ions are not resistant to high temperatures and are easily degraded; positive ions require high temperatures to remove solvents; and at 400 / 350 ℃, the positive and negative modes were optimal respectively, with no thermal degradation and complete solvent removal.

[0087] 7. Selection of desolventizing gas flow rate The following desolventizing gas flow rates were selected in the experiment: 500, 600, and 700 L / h. The results showed that low flow rates resulted in poor water removal; high flow rates consumed more gas and extinguished ions; while 600 L / h was the general optimal balance point.

[0088] 8. Selection of collision airflow velocity The following collision gas flow rates were selected during the experiment: 0.3, 0.5, and 0.7 mL / min. The results showed that the full scan mode did not cause fragmentation, but only maintained vacuum stability. 0.5 mL / min: the instrument's default standard value, which does not require optimization.

[0089] 9. Selection of scanning time The following scan times are available for selection in the experiment: 0.2, 0.3, 0.5, and 1.0 s; The results showed that if the scan time was too fast, the number of points would be insufficient and the quality accuracy would be poor; if the scan time was too slow, the narrow peak sampling of UPLC would be insufficient; when the scan time was 0.5 s, the resolution, sensitivity, and the number of chromatographic peak sampling points were balanced.

[0090] 10. Selection of scanning range The following scan ranges were available for selection in the experiment: 50–1000, 50–1200, and 100–1500; the results showed that 50–1200 was the most common range for non-target metabolomics.

[0091] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing a characteristic map of compound pine tablets, characterized in that, Includes the following steps: a) Preparation of the test solution of compound pine tablets: Weigh the compound pine tablet extract, add methanol-water solution, extract by ultrasonication, centrifuge, take the supernatant, filter, and obtain the test solution; b) The test solution was analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry, and a total ion chromatogram was collected; c) Perform peak extraction, peak alignment, and peak correction on the total ion chromatogram to establish a characteristic spectrum of Fusong tablets; The chromatographic conditions for the ultra-high performance liquid chromatography include: The chromatographic column was a CORTECS T3 column; Mobile phase A is an aqueous solution containing formic acid, and mobile phase B is an acetonitrile solution containing formic acid.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography also include: Column specifications: 100mm × 2.1mm, 1.6μm; Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; Column temperature: 35℃; Sample chamber temperature: 10℃; Flow rate: 0.3 mL / min; Injection volume: 2 μL.

3. The construction method according to claim 1, characterized in that, The mass spectrometry conditions for the ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry system include: The ion source is an electrospray ionization source; Scanning was performed using both positive and negative ion modes. The capillary voltage is 3.0 kV in positive ion mode and 2.3 kV in negative ion mode; The voltage at the conical hole is 40 V; The extraction cone voltage is 3 V; The ion source temperature is 100℃; The desolvation gas temperature is 400℃ in positive ion mode and 350℃ in negative ion mode. The solvent removal gas flow rate is 600 L / h; The collision gas flow rate was 0.5 mL / min; The scan time is 0.5 s; The scan interval is 0.02 s; The mass-to-charge ratio scanning range is 50-1200 m / z; The locked mass numbers are [M+H]+556.2771 and [MH]. 554.2615.

4. The construction method according to claim 1, characterized in that, The characteristic spectrum contains 22 characteristic peaks with the following retention times: Peak 1, sucrose, 0.87 min; Peak 5, citric acid, 1.05 min; Peak 6, isocitrate, 1.41 min; Peak 11, adenine, 2.28 min; Peak 17, neochlorogenic acid, 4.7 min; Peak 18, caffeic acid, 5.06 min; Peak 19, salvianolic acid F, 5.54 min; Peak 25, chlorogenic acid, 6.24 min; Peak 27, cryptochlorogenic acid, 6.65 min; Peak 38, puerarin, 8.28 min; Peak 45, ferulic acid, 10.05 min; Peak 50, daidzein, 10.80 min; Peak 63, verrucoside glucoside, 14.19 min; Peak 76, 3,4-dicaffeoylquinic acid, 17.23 min; Peak 78, ligustrazine F... Peak 89: 3,5-dicaffeoylquinic acid 23.45 min; Peak 94: gentianin 24.2 min; Peak 96: daidzein 24.46 min; Peak 105: naringenin 26.1 min; Peak 115: gentianin 28.65 min; Peak 141: E-ligustilide 37.12 min; Peak 142: n-butylphthalide 37.22 min.

5. The construction method according to claim 4, characterized in that, The feature spectrum also contains 115 characteristic peaks; The relative retention times of the 115 characteristic peaks are as follows: 。 6. A quality control method for pine nut tablets, characterized in that, Includes the following steps: (1) Provide a reference feature spectrum for lysine tablets; the reference feature spectrum is generated by testing multiple batches of qualified lysine tablet samples using the feature spectrum construction method described in any one of claims 1-5; (2) Using the feature map construction method according to any one of claims 1-5, a feature map of the sample of the pine nut tablets to be tested is constructed; (3) Compare the characteristic spectrum of the sample to be tested obtained in step (2) with the control characteristic spectrum obtained in step (1), and determine the quality of the sample to be tested based on the retention time and / or peak area matching of the common peak.

7. The quality control method according to claim 6, characterized in that, The comparison includes matching the characteristic spectrum of the test compound tablet with the characteristic spectrum of one or more control compounds by retention time and / or mass spectrometry information.

8. The quality control method according to claim 7, characterized in that, The control compounds include one or more of the following: sucrose, citric acid, isocitrate, adenine, neochlorogenic acid, caffeic acid, salvianolic acid F, chlorogenic acid, cryptochlorogenic acid, puerarin, ferulic acid, daidzein, 3,4-dicaffeoylquinic acid, ligustilide F, 3,5-dicaffeoylquinic acid, daidzein, daidzein, naringenin, daidzein, E-ligustilide, n-butylphthalide, and verrucoside.

9. The quality control method according to claim 6, characterized in that, The quality assessment includes one or more of the following: finished product appearance inspection, finished product identification inspection, finished product n-butanol extract content inspection, and semi-finished product qualitative inspection.

10. The quality control method according to claim 6, characterized in that, The compounds identified in the characteristic chromatograms are used to screen for potential active ingredients in lysine tablets for the treatment of systemic sclerosis.

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