UPLC-Q-TOF-MS Analysis Method and Application of Taohong Siwu Decoction

Abstract

This invention provides a UPLC-Q-TOF-MS analytical method and application for Taohong Siwu Decoction, belonging to the field of drug detection technology. The analytical method employs ultra-high performance liquid chromatography-tandem mass spectrometry with acetonitrile-formic acid aqueous solution as the mobile phase, using gradient elution to characterize the components in Taohong Siwu Decoction. The characterization results obtained are used for quality evaluation or control throughout the research, development, production, and clinical application of Taohong Siwu Decoction. By adjusting the chromatographic and mass spectrometric conditions, this invention uses UPLC-Q-TOF-MS to detect and analyze Taohong Siwu Decoction, effectively separating and detecting 40 components and qualitatively identifying 38 of them.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a UPLC-Q-TOF-MS analysis method and application for Taohong Siwu Decoction. Background Technology

[0002] Taohong Siwu Decoction is a blood-activating and qi-tonifying formula composed of roasted peach kernel, wine-processed safflower, wine-processed white peony root, wine-processed angelica root, chuanxiong rhizome, and wine-processed rehmannia root. It originates from *Fuke Bingjian* (A Mirror of Gynecology) written by Chai Dehua in the Qing Dynasty and is included in the *Catalogue of Ancient Classic Prescriptions (First Batch)*. In traditional Chinese medicine clinical practice, this formula is used to treat blood deficiency with blood stasis, as well as gynecological conditions such as early menstruation, heavy bleeding with clots, thick and purplish menstrual blood, and abdominal pain. It is widely used for various blood stasis and blood deficiency syndromes. Furthermore, it shows good efficacy in diseases where traditional Chinese medicine has advantages, such as cardiovascular and cerebrovascular diseases, obstetric and gynecological diseases, and orthopedic diseases. Therefore, developing this formula into a classic Chinese medicine compound preparation has good clinical value and application prospects.

[0003] Currently, the quality evaluation standards for traditional Chinese medicine (TCM) suffer from problems such as poor scientific rigor, poor traceability, and poor quality uniformity, which have become a major constraint on the industry's development. In the research and development of classic formula compound preparations, the first step is to conduct "material benchmark" quality research. "Classic formula material benchmarks" refer to the standards for medicinal substances in TCM prepared based on the preparation methods of classic formulas recorded in ancient medical books. Except for the molding process, the other preparation methods should be basically consistent with the ancient records. All pharmaceutical studies of classic formula compound preparations must be compared with the material benchmarks to ensure consistency with the key quality attributes of the material benchmarks. Therefore, establishing a quality control system centered on the identification and value transfer of key quality attributes is the core of developing high-quality classic formula compound preparations.

[0004] The composition of Taohong Siwu Decoction is complex, and the components interfere with each other, making them difficult to separate and detect. Although there are some studies on Taohong Siwu Decoction, the existing analytical methods for examining it can only analyze a limited number of components and are relatively one-sided, failing to comprehensively analyze the main components of Taohong Siwu Decoction. Therefore, further research is needed to determine which other main components Taohong Siwu Decoction contains. Summary of the Invention

[0005] To address the above problems, this invention provides a UPLC-Q-TOF-MS analysis method and application for Taohong Siwu Decoction.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A UPLC-Q-TOF-MS analytical method for Taohong Siwu Decoction, wherein the analytical method uses ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry to characterize the components in Taohong Siwu Decoction;

[0008] The ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry technique uses acetonitrile as mobile phase A and formic acid aqueous solution with a concentration of 0.08–0.12 wt% as mobile phase B.

[0009] The elution method is gradient elution; the elution conditions for the gradient elution are:

[0010] 0–5.50 min, 15% → 20% mobile phase A, 85% → 80% mobile phase B;

[0011] 5.50–8.50 min, 20% → 40% mobile phase A, 80% → 60% mobile phase B;

[0012] 8.50–17.00 min, 40% → 65% mobile phase A, 60% → 35% mobile phase B.

[0013] Furthermore, the analysis method also includes:

[0014] Using single-herb samples of peach kernel, safflower, white peony root, angelica root, chuanxiong rhizome, and rehmannia root, the same chromatographic and mass spectrometric conditions were used to characterize them in order to determine the source of each herb in Taohong Siwu Decoction.

[0015] Furthermore, the analytical method also includes:

[0016] Using paeoniflorin, ligustrolide I, caffeic acid, and ferulic acid as reference standards, the components were characterized under the same chromatographic and mass spectrometric conditions to determine the types of components corresponding to each component in Taohong Siwu Decoction.

[0017] Furthermore, the mass spectrometry detection of the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry technique was performed in both positive and negative ion modes, with a drying gas temperature of 348–352°C, a drying gas flow rate of 9–11 L / min, a nebulizing gas pressure of 34–36 psi, a sheath gas temperature of 348–352°C, a sheath gas flow rate of 11–13 L / min, a capillary voltage of 3900–4100 V in positive mode, and a capillary voltage of 3400–3600 V in negative mode.

[0018] Furthermore, the first-stage mass spectrometry of the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry technique uses MS mode, with a mass scan range of 100–1000 m / z.

[0019] Furthermore, the secondary mass spectrometry of the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry technique uses the Auto-MS / MS mode, with collision voltages of 10V, 20V, and 30V, respectively.

[0020] Furthermore, the chromatographic conditions for the ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry technique are as follows: the chromatographic column is an Ultimate UHPLC XB-C18 column; the flow rate is 0.3–0.5 mL / min; the column temperature is 25–30 °C; and the detection wavelength is 240 nm.

[0021] Furthermore, the analytical method specifically includes the following steps:

[0022] Take Taohong Siwu Decoction and prepare the test solution;

[0023] Take the following herbs separately: peach kernel from the mountain, safflower from wine, white peony root from wine, angelica root from wine, chuanxiong rhizome from wine, and rehmannia root from wine, and prepare corresponding single herb sample solutions;

[0024] Prepare a reference solution by taking paeoniflorin, ligustrazine lactone I, caffeic acid and ferulic acid;

[0025] The test solution, the single herb sample solution and the reference solution were taken separately and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to determine the types of components contained in Taohong Siwu Decoction and the corresponding Chinese medicine sources of each component.

[0026] The analysis involved comparing the chromatographic and mass spectrometric results of the test sample solution with those of each single herb sample solution and the reference solution, and comparing them with literature data to determine the types of components in Taohong Siwu Decoction and the corresponding Chinese herbal medicine sources.

[0027] The analysis can also be performed by comparing the chromatographic and mass spectrometric results of the test sample obtained from the test sample solution with the information recorded in Table 1 of this invention to determine the types of ingredients contained in Taohong Siwu Decoction and the corresponding Chinese medicine sources of each ingredient.

[0028] The chromatographic results are UPLC-UV chromatographic results, that is, chromatograms or information in chromatograms obtained by ultra-high performance liquid chromatography in tandem quadrupole time-of-flight mass spectrometry; the information in chromatograms includes peak numbers and corresponding retention times, etc.

[0029] The mass spectrometry results are the total ion current results in UPLC-MS positive and negative modes, that is, the total ion current chromatograms in positive and negative modes obtained by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry, or the information in the total ion current chromatograms in positive and negative modes; the information in the total ion current chromatograms in positive and negative modes includes information such as adduct ions in the first-level positive and negative modes, characteristic fragment ions in the second-level positive or negative modes, measured molecular weight, and theoretical molecular weight.

[0030] Furthermore, the test solution is prepared by dissolving Taohong Siwu Decoction in water, extracting with ethyl acetate, separating the phases, evaporating the obtained ethyl acetate solution to dryness, and then dissolving it in methanol.

[0031] Each single herb sample solution was prepared by separately decocting the following herbs in water: peach kernel, safflower, white peony root, angelica root, chuanxiong rhizome, and rehmannia root. The resulting filtrates were extracted with ethyl acetate, separated, and the ethyl acetate solution was evaporated to dryness and then dissolved in methanol.

[0032] The characterization results of the components in Taohong Siwu Decoction obtained using the above analytical methods are applied in the quality evaluation or control of the entire process of research / development / production / clinical application of Taohong Siwu Decoction. The characterization results show that 40 chromatographic peaks were separated and detected in Taohong Siwu Decoction, and 38 chromatographic peaks were identified, namely: Peak 1 is gallic acid, Peak 2 is 5-hydroxymethylfurfural, Peak 3 is p-hydroxybenzoic acid, Peak 4 is caffeic acid, Peak 5 is paeoniflorin, Peak 6 is peoniflorin, Peak 7 is paeoniflorin, Peak 8 is paeonolide I, Peak 9 is coumaric acid, and Peak 10 is 6-Hydroxykaempferol-3-O- β-D-glucoside, peak 11 is 3-hydroxy-4,5,6,7-tetrahydro-6,7-dihydroxy-3-butylphthalide, peak 12 is ferulic acid, peak 13 is rehmannia violetin A, peak 14 is 6-O-Galloylalbiflorin, peak 15 is galloylpaeoniflorin, peak 16 is verbascoside, peak 17 is chuanxionolide J, peak 18 is Kaempferol-3-O-β-rutinoside, and peak 19 is chuanxiongnolide. Peaks R1 and 20 are ligustilide I, peak 21 is paeoniflorin, peak 22 is ligustilide H, peak 23 is artemisia argyi, peak 24 is 2Z-decaene-4,6-diyn-1-OZD-glucopyranoside, peak 25 is N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine, peak 26 is N(1),N(5),N(10)-(E)-tri-p-coumaroylspermidine, and peak 27 is Safflospermidine. A. Peak 28 is benzoylpaeoniflorin; Peak 29 is paeoniflorin R3; Peak 30 is ferulic acid coniferyl ester; Peak 31 is ligustilide A; Peak 32 is ligustilide F; Peak 33 is ligustilide G; Peak 36 is ligustilide B; Peak 37 is ligustilide A; Peak 38 is E-ligustilide; Peak 39 is Z-ligustilide; Peak 40 is Chuanxiongnolide L2.

[0033] The characterization results of the components are the types of components contained in Taohong Siwu Decoction and the corresponding Chinese medicine sources of each component.

[0034] The beneficial effects of the UPLC-Q-TOF-MS analysis method and its application for Taohong Siwu Decoction of the present invention are as follows:

[0035] This invention, by adjusting the elution process, successfully separated and detected 40 components in Taohong Siwu Decoction, especially those that cannot be separated and detected in a single step, such as gallic acid, 5-hydroxymethylfurfural, wild cheriin, 6-Hydroxykaempferol-3-O-β-D-glucoside, 3-hydroxy-4,5,6,7-tetrahydro-6,7-dihydroxy-3-butylphthalide, rehmannia violetin A, 6-O-Galloylalbiflorin, chuanxiong lactone J, Kaempferol-3-O-β-rutinoside, and chuanxiongnolide. R1, Ligusticum striatum lactone I, Ligusticum striatum lactone H, Artemisia capillaris, 2Z-decaene-4,6-diyn-1-OZD-glucopyranoside, N(1), N(5), N(10)-(Z)-tri-p-coumaroylspermidine, N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine, Safflospermidine A, Benzoylpaeoniflorin, Paeoniflorin R3, Ferulic acid coniferyl alcohol ester, Ligusticum striatum lactone A, Ligusticum striatum lactone F, Ligusticum striatum lactone G, Ligusticum striatum lactone B, E-Ligusticum striatum lactone, Z-Ligusticum striatum lactone and Chuanxiongnolide L2, etc.; The analytical method of this invention can successfully separate, detect and characterize these components in a single injection, providing more sufficient scientific basis for the research of Taohong Siwu Decoction;

[0036] This invention, by adjusting chromatographic and mass spectrometric conditions, uses UPLC-Q-TOF-MS to detect and analyze Taohong Siwu Decoction, effectively separating and detecting 40 components in Taohong Siwu Decoction, and qualitatively identifying 38 of them.

[0037] This invention employs the UPLC-Q-TOF-MS analytical method, which significantly improves detection efficiency. By using ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry for a single analysis, 40 chemical components in Taohong Siwu Decoction can be successfully analyzed, and 38 components can be identified. Using these 40 components (including the 38 identified components) as detection indicators in this invention is more conducive to assessing the pharmacological activity and safety of Taohong Siwu Decoction.

[0038] This invention employs the UPLC-Q-TOF-MS analysis method, which is highly efficient and fast, and can obtain accurate molecular structure information. It can obtain first-level and multi-level mass spectrometry information of each compound, providing a good technical means for the analysis of multiple components in Taohong Siwu Decoction, with extremely high sensitivity and excellent qualitative ability.

[0039] The 40 chemical components in Taohong Siwu Decoction analyzed in this invention have subtle structural differences, and the resulting mass spectrometry feature fragments have significant differences, which can accurately identify the active ingredients.

[0040] This invention uses the UPLC-Q-TOF-MS analysis method. Only the construction of the mass spectrum library needs to be completed on the instrument. In the subsequent analysis process, no reference standard is required. Only the sample needs to be tested. The sample test results are compared with the mass spectrum library to confirm whether the sample contains the target active ingredient.

[0041] The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction of the present invention is applicable to the detection of active ingredients in various Taohong Siwu Decoction preparations, thereby determining the pharmacological activity of Taohong Siwu Decoction;

[0042] The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction of the present invention is simple, accurate, stable, reproducible, and has good separation effect of active ingredients. It can be used as an important method for examining the pharmacological activity of Taohong Siwu Decoction. Attached Figure Description

[0043] Figure 1 These are the UPLC-MS-TIC and UPLC-UV chromatograms of the test solution in positive and negative modes in Example 1 of this invention, wherein... Figure 1 The top-middle image shows the UPLC-MS-TIC chromatogram in positive mode, the middle image shows the UPLC-MS-TIC chromatogram in positive mode, and the bottom image shows the UPLC-UV chromatogram (240nm). Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0045] Example 1: A UPLC-Q-TOF-MS analysis method and application of Taohong Siwu Decoction

[0046] In this embodiment, the Taohong Siwu Decoction reference sample is used as the test sample. The preparation method of the Taohong Siwu Decoction reference sample is as follows:

[0047] Prescription: Rehmannia glutinosa (processed with wine) 11.19g, Angelica sinensis (processed with wine) 14.92g

[0048] White peony root (processed with wine) 5.60g, Ligusticum chuanxiong 3.73g

[0049] 3.78g of roasted peach kernels and 3.73g of safflower.

[0050] Preparation: Grind the above six ingredients, including the peach kernels from the mountain, into a paste. Place the paste and the remaining five ingredients in a clay pot and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat, and simmer over low heat for 30 minutes. Filter. For the second decoction, add 8 times the amount of water, bring to a boil over high heat, and simmer over low heat for 20 minutes. Filter. Combine the above filtrates and concentrate under reduced pressure until the ratio of the amount of medicinal slices to the volume of the concentrated liquid is approximately 1:2 (g:ml). Freeze-dry and pulverize to obtain the reference sample.

[0051] In this embodiment, the chemical composition of the Taohong Siwu Decoction reference sample was qualitatively analyzed using the UPLC-Q-TOF-MS method. The specific analytical method is as follows:

[0052] 1) Preparation of test solution: Weigh 1g of Taohong Siwu Decoction reference sample accurately, place it in a stoppered conical flask, add 25mL of water accurately, sonicate to dissolve it, extract with ethyl acetate three times by shaking, each time using 25mL of ethyl acetate, separate the phases, combine to obtain ethyl acetate solution, evaporate to dryness, dissolve the residue with an appropriate amount of methanol, transfer to a 10mL volumetric flask, dilute with methanol to the mark, shake well, filter, and the resulting filtrate is the test solution.

[0053] Preparation of single-herb sample solutions: Accurately weigh 0.2g of *Prunus persica* kernel powder (passed through a No. 3 sieve), 0.2g of *Carthamus tinctorius* (passed through a No. 3 sieve), 0.3g of *Paeonia lactiflora* (passed through a No. 3 sieve), 0.8g of *Angelica sinensis* (passed through a No. 3 sieve), 0.2g of *Ligusticum chuanxiong* (passed through a No. 3 sieve), and 0.6g of *Rehmannia glutinosa* (passed through a No. 3 sieve). Place them in round-bottom flasks, add 50mL of water, decoct for 50 minutes, filter, and extract the corresponding filtrates three times with 25mL of ethyl acetate each time. Separate the phases, combine the filtrates to obtain the corresponding ethyl acetate solutions, evaporate to dryness, dissolve the residue in an appropriate amount of methanol, transfer to a 10mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrates to obtain the sample solutions of each single herb.

[0054] Preparation of reference solutions: Take appropriate amounts of paeoniflorin, ligustrol I, caffeic acid and ferulic acid reference standards, accurately weigh them, and add 70% methanol to prepare solutions containing 50 μg paeoniflorin, 40 μg ligustrol I, 50 μg caffeic acid and 80 μg ferulic acid per 1 mL, respectively, to obtain the reference solutions.

[0055] 2) Take the test sample solution, the sample solutions of each single herb, and the reference solution respectively and perform ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (i.e., UPLC-Q-TOF-MS) to obtain the chromatographic and mass spectrometric results of the test sample, the chromatographic and mass spectrometric results of each single herb, and the chromatographic and mass spectrometric results of the reference.

[0056] The chromatographic results are UPLC-UV chromatographic results, that is, chromatograms or information in chromatograms obtained by ultra-high performance liquid chromatography in tandem quadrupole time-of-flight mass spectrometry; the information in chromatograms includes peak numbers and corresponding retention times, etc.

[0057] The mass spectrometry results are the total ion current results in UPLC-MS positive and negative modes, that is, the total ion current chromatograms in positive and negative modes obtained by ultra-high performance liquid chromatography in tandem quadrupole time-of-flight mass spectrometry, or the information in the total ion current chromatograms in positive and negative modes; the information in the total ion current chromatograms in positive and negative modes includes information such as adduct ions in the first-level positive and negative modes, characteristic fragment ions in the second-level positive or negative modes, measured molecular weight, and theoretical molecular weight.

[0058] The chromatographic and mass spectrometric results of the test sample were compared and analyzed with the chromatographic and mass spectrometric results of each single herb to determine the source of each Chinese medicine corresponding to each component detected in Taohong Siwu Decoction.

[0059] The chromatographic and mass spectrometric results of the test sample were compared and analyzed with those of the reference sample to determine the types of some components detected in Taohong Siwu Decoction.

[0060] The instrument used is an Agilent 1290II ultra-high performance liquid chromatography (UPLC) system connected to a G6530C quadrupole-time-of-flight tandem mass spectrometer (Q-TOF-MS), equipped with an independent quaternary pump, autosampler, column oven, diode array detector (DAD), and electrospray ionization source (ESI).

[0061] The chromatographic conditions are as follows:

[0062] Chromatographic separation was performed using an Ultimate UHPLC XB-C18 column (2.1×100mm, 1.8μm) equipped with an online filter;

[0063] The flow rate was 0.4 mL / min;

[0064] The column temperature is 30℃;

[0065] The detection wavelength is 240nm;

[0066] The injection volume was 1 μL;

[0067] Mobile phase A is acetonitrile, and mobile phase B is a 0.1 wt% aqueous formic acid solution;

[0068] The elution method is gradient elution, and the specific elution procedure is as follows:

[0069] 0–5.50 min, 15% → 20% mobile phase A, 85% → 80% mobile phase B;

[0070] 5.50–8.50 min, 20% → 40% mobile phase A, 80% → 60% mobile phase B;

[0071] 8.50–17.00 min, 40% → 65% mobile phase A, 60% → 35% mobile phase B.

[0072] Mass spectrometry conditions are:

[0073] Mass spectrometry detection was performed in positive and negative ion modes, with a drying gas temperature of 350℃, a drying gas flow rate of 10 L / min, a nebulizing gas pressure of 35 psi, a sheath gas temperature of 350℃, a sheath gas flow rate of 12 L / min, and capillary voltages of 4000 V (positive mode) and 3500 V (negative mode), respectively.

[0074] The first-stage mass spectrometer was set to MS mode with a mass scan range of 100–1000 m / z.

[0075] The secondary mass spectrometer was set to Auto-MS / MS mode with collision voltages of 10V, 20V and 30V.

[0076] The data was collected using Agilent MassHunter (B.08.00) software, and data processing was performed using Agilent Qualitative Navigator (B.08.00) and Qualitative Workflows (B.08.00) software.

[0077] Analysis was performed using UPLC-UV chromatograms and UPLC-MS total ion chromatograms in both positive and negative modes of the test sample solution. (See attached figures.) Figure 1 ,in Figure 1 The top-middle image shows the UPLC-MS-TIC chromatogram in positive mode; the middle image shows the UPLC-MS-TIC chromatogram in positive mode; and the bottom image shows the UPLC-UV chromatogram (240 nm). Figure 1 The substances in chromatographic peaks 1 to 40 marked in the figure were all well separated and detected.

[0078] By comparing and analyzing the mass spectrometry results of the test sample solution with those of individual samples of six medicinal herbs (rehmannia root, angelica root, chuanxiong rhizome, white peony root, peach kernel, and safflower), the source of 40 chromatographic peaks was identified, and the main chromatographic peaks were characterized.

[0079] The mass spectrometry analysis results are as follows:

[0080] a. Primary mass spectrometry analysis

[0081] Accurate mass number determination by high-resolution TOF-MS was performed on... Figure 1 The compounds in peaks 1-40 were analyzed, and the results are shown in Table 1. Table 1 shows that these compounds, in positive mode, can generate the molecular ion peak [M+H]. + [M+Na] + and [M+NH4] + In isoadductor mode, a molecular ion peak [MH] can be generated; however, in negative mode, a molecular ion peak can be generated. - [M+Cl] - and [M+HCOO] - Isoadduct ions. Based on this information about adduct ions, the precise molecular weight and molecular formula of the compound can be accurately deduced (mass measurement error ≤ 5 ppm, indicating correct matching results), thus aiding in subsequent structural identification.

[0082] b. Secondary mass spectrometry analysis

[0083] The test solution was analyzed by Auto-MS / MS under the same chromatographic conditions, and the structures of peaks 1 to 40 were confirmed. The results are shown in Table 1.

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] UPLC-Q-TOF-MS analysis of the Taohong Siwu Decoction reference sample detected 40 components. By comparing with literature data, comparing retention times with reference standards, and analyzing the accurate molecular weights provided by UPLC-Q-TOF-MS, 38 components were identified, including:

[0096] ① Monoterpenoid glycosides

[0097] Analysis of the Taohong Siwu Decoction reference sample identified nine monoterpenoid glycosides, namely paeoniflorin, paeoniflorin, galloylpaeoniflorin, paeonioside I, 6-O-Galloylalbiflorin, paeoniflorin, benzoylpaeoniflorin, paeoniflorin R3, and rehmannia violetin A. Except for rehmannia violetin A, all nine monoterpenoid glycosides are exclusive components of wine-processed white peony root.

[0098] Among them, paeoniflorin was compared with the results of the reference standard and its accurate chemical structure was confirmed.

[0099] ②Phenylphthalide compounds

[0100] Analysis of the Taohong Siwu Decoction reference sample identified 13 phthalide compounds, namely 3-hydroxy-4,5,6,7-tetrahydro-6,7-dihydroxy-3-butylphthalide, chuanxiong lactone J, chuanxiongnolide R1, chuanxiong lactone I, chuanxiong lactone H, chuanxiong lactone A, chuanxiong lactone F, chuanxiong lactone G, chuanxiong lactone B, chuanxiong lactone A, E-ligustilide, Z-ligustilide, and chuanxiongnolide L2. Except for 3-hydroxy-4,5,6,7-tetrahydro-6,7-dihydroxy-3-butylphthalide, which is unique to Ligusticum chuanxiong, the other phthalide compounds are common to both Ligusticum chuanxiong and Angelica sinensis.

[0101] Among them, the accurate chemical structure of Ligusticum lactone I was confirmed by comparing it with the results of the reference standard.

[0102] ③ Spermine compounds

[0103] Analysis of the Taohong Siwu Decoction reference sample identified three spermidine compounds: N(1), N(5), N(10)-(Z)-tri-p-coumaroylsper-midine, N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine, and Safflospermidine A. All three spermidine compounds are exclusive components of safflower.

[0104] ④ Phenolic acids

[0105] Analysis of the Taohong Siwu Decoction reference sample identified five phenolic acid compounds: gallic acid, p-hydroxybenzoic acid, caffeic acid, ferulic acid, and coumaric acid. Gallic acid is a specific component of white peony root (processed with wine), p-hydroxybenzoic acid is a shared component of angelica root (processed with wine) and safflower (processed with wine), caffeic acid is a shared component of angelica root (processed with wine), chuanxiong rhizome (processed with wine), and safflower, ferulic acid is a shared component of angelica root (processed with wine) and chuanxiong rhizome (processed with wine), and coumaric acid is a specific component of safflower.

[0106] The accurate chemical structures of both caffeic acid and ferulic acid were confirmed by comparing them with the results of reference standards.

[0107] ⑤ Flavonoids

[0108] Analysis of the Taohong Siwu Decoction reference sample identified two flavonoids: 6-Hydroxykaempferol-3-O-β-D-glucoside and Kaempferol-3-O-β-rutinoside. Both flavonoids are components of safflower.

[0109] ⑥ Other categories

[0110] Analysis of the reference sample of Taohong Siwu Decoction identified six other compounds: safflower glycoside, 5-hydroxymethylfurfural, verbascoside, artemisia argyi, 2Z-decaene-4,6-diyn-1-OZD-glucopyranoside, and ferulic acid coniferyl ester. Safflower glycoside is a proprietary component of *Prunus persica* kernel, 5-hydroxymethylfurfural and artemisia argyi are proprietary components of *Angelica sinensis*, verbascoside is a proprietary component of *Rehmannia glutinosa*, and 2Z-decaene-4,6-diyn-1-OZD-glucopyranoside is a proprietary component of *Carthamus tinctorius*.

[0111] In summary, the qualitative study results of the chemical composition in the Taohong Siwu Decoction reference sample are shown in Table 2.

[0112] Table 2. Summary of Qualitative Study Results of Chemical Components in Taohong Siwu Decoction Reference Samples

[0113]

[0114]

[0115] Based on a review of chemical literature, we analyzed and deduced the components such as monoterpenoid glycosides, phenols, spermidines, phenolic acids, and flavonoids present in the reference sample of Taohong Siwu Decoction. We then used high-resolution UPLC-Q-TOF-MS to identify the specific compounds corresponding to 38 components, of which 4 components were confirmed by comparison with reference standards.

[0116] In the subsequent application of Taohong Siwu Decoction in the entire process of quality evaluation or control during research, development, production, and clinical application, the above-mentioned detection method is used to test the Taohong Siwu Decoction to be tested. The obtained chromatographic and mass spectrometric results of the Taohong Siwu Decoction to be tested are compared with the retention time, measured molecular weight, theoretical molecular weight, and other information recorded in Table 1 (i.e., the types of components contained in Taohong Siwu Decoction obtained in this invention and the corresponding Chinese herbal medicine sources), to determine the types of components contained in the Taohong Siwu Decoction to be tested and the corresponding Chinese herbal medicine sources, thereby judging the quality of the Taohong Siwu Decoction to be tested. When one or more components are obviously missing in the Taohong Siwu Decoction to be tested, it indicates that the quality of the Taohong Siwu Decoction to be tested does not meet the standards.

[0117] UPLC-Q-TOF-MS Analysis Method for Taohong Siwu Decoction in Examples 2-3

[0118] Examples 2 and 3 are UPLC-Q-TOF-MS analytical methods for Taohong Siwu Decoction. The steps are basically the same as those in Example 1, with the only difference being some parameters. See Table 2 for details.

[0119] Table 2. Summary of parameters in Examples 2-3

[0120]

[0121] The contents of other parts of Examples 2 and 3 are the same as those of Example 1, and the analysis results are also basically the same as those of Example 1, so they will not be repeated here.

[0122] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A UPLC-Q-TOF-MS analytical method for Taohong Siwu Decoction, characterized in that, The analytical method is to take Taohong Siwu Decoction, dissolve it in water, extract it with ethyl acetate, separate the phases, evaporate the ethyl acetate solution to dryness, dissolve it in methanol to prepare the test solution, and characterize the components in Taohong Siwu Decoction using a single-pass ultra-high performance liquid chromatography-tandem mass spectrometry technique. The ultra-high performance liquid chromatography-tandem mass spectrometry technique uses acetonitrile as mobile phase A and formic acid aqueous solution with a concentration of 0.08~0.12wt% as mobile phase B. The elution method is gradient elution; the elution conditions for the gradient elution are: 0~5.50 min, 15%→20% mobile phase A, 85%→80% mobile phase B; 5.50~8.50 min, 20%→40% mobile phase A, 80%→60% mobile phase B; 8.50~17.00 min, 40%→65% mobile phase A, 60%→35% mobile phase B; The chromatographic column was an Ultimate UHPLC XB-C18 column, 2.1×100mm, 1.8μm; The mass spectrometry detection of the ultra-high performance liquid chromatography-tandem mass spectrometry technique uses an electrospray ionization source, and is performed in positive and negative ion modes respectively. The drying gas temperature is 348~352℃, the drying gas flow rate is 9~11L / min, the nebulizing gas pressure is 34~36psi, the sheath gas temperature is 348~352℃, the sheath gas flow rate is 11~13L / min, the capillary voltage is 3900~4100V in positive mode, and the capillary voltage is 3400~3600V in negative mode. The characterization results of the components showed that 40 chromatographic peaks were separated and detected in Taohong Siwu Decoction, and 38 chromatographic peaks were identified as follows: Peak 1 was gallic acid, Peak 2 was 5-hydroxymethylfurfural, Peak 3 was p-hydroxybenzoic acid, Peak 4 was caffeic acid, Peak 5 was paeoniflorin, Peak 6 was safflower glycoside, Peak 7 was paeoniflorin, Peak 8 was paeonolide I, Peak 9 was coumaric acid, and Peak 10 was 6-Hydroxykaempferol-3-O-β-D-glucoside. Peak 11 is 3-hydroxy-4,5,6,7-tetrahydro-6,7-dihydroxy-3-butylphthalide; peak 12 is ferulic acid; peak 13 is rehmannia violetin A; peak 14 is 6-O-Galloylalbiflorin; peak 15 is galloylpaeoniflorin; peak 16 is verbascoside; peak 17 is ligustrol J; and peak 18 is Kaempferol-3-O-β- Peak 19 is rutinoside, peak 20 is chuanxiongnolide R1, peak 21 is paeoniflorin, peak 22 is chuanxiongnolide H, peak 23 is artemisia argyi, peak 24 is 2Z-decaene-4,6-diyn-1-OZD-glucopyranoside, peak 25 is N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine, peak 26 is N(1),N(5),N(10)-(E)-tri-p-coumaroylspermidine, and peak 27 is Safflospermidine. A. Peak 28 is benzoylpaeoniflorin, peak 29 is paeoniflorin R3, peak 30 is ferulic acid coniferyl ester, peak 31 is chuanxiong lactone A, peak 32 is chuanxiong lactone F, peak 33 is chuanxiong lactone G, peak 36 is chuanxiong lactone B, peak 37 is chuanxiong lactone A, peak 38 is E-ligustilide, peak 39 is Z-ligustilide, and peak 40 is chuanxiongnolide L2.

2. The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction according to claim 1, characterized in that, The analytical method further includes: Using single-herb samples of peach kernel, safflower, white peony root, angelica root, chuanxiong rhizome, and rehmannia root, and characterized under the same chromatographic and mass spectrometric conditions, the source of each component in Taohong Siwu Decoction was determined.

3. The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction according to claim 1 or 2, characterized in that, The analytical methods also include: Using paeoniflorin, ligustrolide I, caffeic acid, and ferulic acid as reference standards, the same chromatographic and mass spectrometric conditions were used to characterize the components in Taohong Siwu Decoction to determine the corresponding component types.

4. The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction according to claim 1 or 2, characterized in that, The first-stage mass spectrometry of the ultra-high performance liquid chromatography-tandem mass spectrometry technique uses MS mode with a mass scan range of 100~1000 m / z.

5. The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction according to claim 1 or 2, characterized in that, The secondary mass spectrometry of the ultra-high performance liquid chromatography-tandem mass spectrometry technique uses the Auto-MS / MS mode, with collision voltages of 10V, 20V and 30V respectively.

6. The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction according to claim 1 or 2, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry technique are as follows: flow rate of 0.3~0.5 mL / min; column temperature of 25~30℃; and detection wavelength of 240 nm.

7. The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction according to claim 1 or 2, characterized in that, The analytical method specifically includes the following steps: Take the following herbs separately: peach kernel from the mountain, safflower from wine, white peony root from wine, angelica root from wine, chuanxiong rhizome from wine, and rehmannia root from wine, and prepare corresponding single herb sample solutions; Prepare a reference solution by taking paeoniflorin, ligustrolide I, caffeic acid and ferulic acid; The test solution, individual herb sample solutions, and reference solutions were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to determine the types of components in Taohong Siwu Decoction and the corresponding Chinese herbal medicine sources.

8. The UPLC-Q-TOF-MS analysis method for Taohong Siwu Decoction according to claim 7, characterized in that, Each single herb sample solution was prepared by separately decocting the following herbs in water: peach kernel, safflower, white peony root, angelica root, chuanxiong rhizome, and rehmannia root. The resulting filtrates were extracted with ethyl acetate, separated, and the ethyl acetate solution was evaporated to dryness and then dissolved in methanol.

9. The application of the characterization results of the components in Taohong Siwu Decoction obtained by the analytical method according to any one of claims 1-8 in the quality evaluation or control of the entire process of research / development / production / clinical application of Taohong Siwu Decoction, characterized in that, The characterization results of the components showed that 40 chromatographic peaks were separated and detected in Taohong Siwu Decoction, and 38 chromatographic peaks were identified as follows: Peak 1 was gallic acid, Peak 2 was 5-hydroxymethylfurfural, Peak 3 was p-hydroxybenzoic acid, Peak 4 was caffeic acid, Peak 5 was paeoniflorin, Peak 6 was safflower glycoside, Peak 7 was paeoniflorin, Peak 8 was paeonolide I, Peak 9 was coumaric acid, and Peak 10 was 6-Hydroxykaempferol-3-O-β-D-glucoside. Peak 11 is 3-hydroxy-4,5,6,7-tetrahydro-6,7-dihydroxy-3-butylphthalide; peak 12 is ferulic acid; peak 13 is rehmannia violetin A; peak 14 is 6-O-Galloylalbiflorin; peak 15 is galloylpaeoniflorin; peak 16 is verbascoside; peak 17 is ligustrol J; and peak 18 is Kaempferol-3-O-β- Peak 19 is rutinoside, peak 20 is chuanxiongnolide R1, peak 21 is paeoniflorin, peak 22 is chuanxiongnolide H, peak 23 is artemisia argyi, peak 24 is 2Z-decaene-4,6-diyn-1-OZD-glucopyranoside, peak 25 is N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine, peak 26 is N(1),N(5),N(10)-(E)-tri-p-coumaroylspermidine, and peak 27 is Safflospermidine. A. Peak 28 is benzoylpaeoniflorin, peak 29 is paeoniflorin R3, peak 30 is ferulic acid coniferyl ester, peak 31 is chuanxiong lactone A, peak 32 is chuanxiong lactone F, peak 33 is chuanxiong lactone G, peak 36 is chuanxiong lactone B, peak 37 is chuanxiong lactone A, peak 38 is E-ligustilide, peak 39 is Z-ligustilide, and peak 40 is chuanxiongnolide L2.

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