A method for constructing the fingerprint of Gualou Xiebai Banxia Decoction and determining the contents of index components

Abstract

The present invention relates to a method for constructing a fingerprint of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction and determining the content of index components, which includes: preparing a test solution from the pharmaceutical preparation of Gualou Xiebai Banxia Decoction; preparing a first mixed reference substance solution from loliolide, chrysoeriol, 25S-Timosaponin BⅡ, Macrostemonoside I, 25R-Timosaponin B II, PWVPG and AVGTNHLLSGETLD; injecting the test solution and the first mixed reference substance solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining the fingerprint of the pharmaceutical preparation according to the liquid chromatography-mass spectrometry method, and establishing a UHPLC-QQQ-MS quantitative detection method. Through the method for constructing the fingerprint of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction and the method for determining the content of index components provided by the present invention, the evaluation standard of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction is comprehensively improved, and the effectiveness of the clinical use of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality analysis and detection of traditional Chinese medicine compound preparations, and particularly relates to a method for constructing a fingerprint of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction and determining the content of index components. Background Art

[0002] Gualou Xiebai Banxia Decoction comes from "Synopsis of the Golden Chamber" written by Zhang Zhongjing in the Han Dynasty and is one of the classic famous prescriptions in the "Catalogue of Ancient Classic Famous Prescriptions" announced by the State Administration of Traditional Chinese Medicine. Its formula consists of Trichosanthes kirilowii Maxim., Allium macrostemon Bunge, Pinellia ternata (Thunb.) Breit., and yellow rice wine, and is mainly used for treating "chest impediment syndrome with phlegm turbidity congestion, manifested as chest pain radiating to the back, inability to lie down comfortably, wheezing, coughing, expectorating, and shortness of breath". It is commonly used in modern times to treat coronary heart disease, angina pectoris, myocardial infarction, chronic bronchitis, pulmonary emphysema, rheumatic heart disease, chronic cholecystitis, etc.

[0003] Since the components of the raw material herbs in Gualou Xiebai Banxia Decoction are relatively complex, and the production areas, ecological environments, harvesting seasons, processing methods, and storage conditions of each Chinese medicinal material are different, there will be great differences in their chemical components and qualities. Therefore, there are often large differences in the composition and content of the decoctions from different production areas and different manufacturers, resulting in unstable quality of Gualou Xiebai Banxia Decoction. So far, the research on the quality control of Gualou Xiebai Banxia Decoction is still relatively weak, which directly affects the development of pharmaceutical preparations of Gualou Xiebai Banxia Decoction.

[0004] Therefore, there is an urgent need to develop a method for constructing a fingerprint of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction and determining the content of index components for use in the quality evaluation of Gualou Xiebai Banxia Decoction. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, the present invention provides a method for constructing a fingerprint of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction and determining the content of index components.

[0006] According to one aspect of the present invention, there is provided a method for constructing a fingerprint of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction, including: preparing a test solution from the pharmaceutical preparation of Gualou Xiebai Banxia Decoction; preparing a first mixed reference solution from loliolide, chrysoeriol, 25 S -Timosaponin B Ⅱ, Macrostemonoside I, 25 R -Timosaponin B II, pentapeptide PWVPG, and tetrapeptide AVGTNHLLSGETLD; injecting the test solution and the first mixed reference solution into a liquid chromatography - mass spectrometry instrument respectively, and obtaining the fingerprint of the pharmaceutical preparation according to the liquid chromatography - mass spectrometry method.

[0007] Preferably, the chromatographic conditions for the liquid chromatography-mass spectrometry method include: stationary phase: a chromatographic column filled with octadecylsilyl-bonded silica gel; mobile phase: mobile phase A is water containing 0.1% (v / v) formic acid, and mobile phase B is an acetonitrile solution containing 0.1% (v / v) formic acid; gradient elution is adopted.

[0008] Preferably, the mass spectrometry conditions for the liquid chromatography-mass spectrometry method include: the ion source is ESI; the backflush nitrogen temperature is 325 °C; the backflush nitrogen flow rate is 11.0 L / min; the nebulizing gas flow rate is 45 psig; the auxiliary nitrogen temperature is 300 °C; the auxiliary nitrogen flow rate is 11.0 L / min; the capillary inlet voltage is 3500 V; the Agilent Jet Stream outlet voltage in the positive ion mode is 500 V (+), and in the negative ion mode is 1500 V (-); the in-source collision voltage is 175 V; the cone voltage is 65.0 V; the Octopole RF Peak is 750 V; the scan mode is full scan; the scan range is m / z 60 - 1700.

[0009] Preferably, the chromatographic column is a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm) chromatographic column.

[0010] Preferably, the gradient elution is carried out according to the following procedure: from 0 to 0.5 min, mobile phase A changes from 85% → 85%, and mobile phase B changes from 15% → 15%; from 0.5 to 3.8 min, mobile phase A changes from 85% → 81%, and mobile phase B changes from 15% → 19%; from 3.8 to 4.5 min, mobile phase A changes from 81% → 77%, and mobile phase B changes from 19% → 23%; from 4.5 to 10.5 min, mobile phase A changes from 77% → 77%, and mobile phase B changes from 23% → 23%; from 10.5 to 12 min, mobile phase A changes from 77% → 69%, and mobile phase B changes from 23% → 31%; from 12 to 16 min, mobile phase A changes from 69% → 64%, and mobile phase B changes from 31% → 36%; from 16 to 16.5 min, mobile phase A changes from 64% → 5%, and mobile phase B changes from 36% → 95%; from 16.5 to 18.9 min, mobile phase A changes from 5% → 5%, and mobile phase B changes from 95% → 95%; from 18.9 to 19 min, mobile phase A changes from 5% → 85%, and mobile phase B changes from 95% → 15%.

[0011] Preferably, in the positive ion mode, the fingerprint contains 12 characteristic peaks, and the numbers of the characteristic peaks are Peak 1A - Peak 12A; the identification of the characteristic peaks is as follows:

[0012]

[0013] Preferably, in the negative ion mode, the fingerprint contains 11 characteristic peaks, numbered from peak 1B to peak 11B; the characteristic peaks and their identifications are as follows:

[0014]

[0015] Preferably, taking peak 8A as the S peak, the relative retention time ranges of peaks 1A to 7A and peaks 9A to 12A relative to the S peak are respectively: 0.328 - 0.334, 0.367 - 0.372, 0.406 - 0.413, 0.494 - 0.499, 0.545 - 0.552, 0.667 - 0.673, 0.738 - 0.744, 1.034 - 1.036, 1.221 - 1.226, 1.637 - 1.649, 1.787 - 1.804.

[0016] Preferably, taking peak 8A as the S peak, the relative standard deviation (RSD) values of the relative retention times of peaks 1A to 7A and peaks 9A to 12A relative to the S peak are respectively: 0.43%, 0.44%, 0.63%, 0.29%, 0.33%, 0.23%, 0.27%, 0.06%, 0.10%, 0.20%, 0.26%.

[0017] Preferably, taking peak 6B as the S peak, the relative retention time ranges of peaks 1B to 5B and peaks 7B to 11B relative to the S peak are respectively: 0.250 - 0.254, 0.555 - 0.561, 0.699 - 0.729, 0.723 - 0.729, 0.844 - 0.850, 1.035 - 1.039, 1.194 - 1.199, 1.248 - 1.255, 1.613 - 1.622, 1.803 - 1.817.

[0018] Preferably, taking peak 6B as the S peak, the relative standard deviation (RSD) values of the relative retention times of peaks 1B to 5B and peaks 7B to 11B relative to the S peak are respectively: 0.47%, 0.27%, 0.21%, 0.21%, 0.19%, 0.09%, 0.12%, 0.16%, 0.16% and 0.22%.

[0019] Preferably, the preparation method of the test solution includes: adding 2000 mL of yellow rice wine to Trichosanthes kirilowii Maxim., Allium macrostemon Bunge and Pinellia ternata (Thunb.) Breit. var. purpurea (Makino) Hsiao et K. C. Hsia, decocting and concentrating to 800 mL, and filtering to obtain the decoction; concentrating the decoction under reduced pressure to obtain a thick extract; treating the diluted thick extract with a solid phase extraction (SPE) column to obtain an eluate; redissolving the dried eluate to a certain concentration to obtain the test solution.

[0020] Preferably, the solid phase extraction (SPE) column is a hydrophilic - lipophilic balance column (HLB).

[0021] Preferably, the treatment of the diluted thick extract using a solid phase extraction (SPE) column includes: sequentially activating the solid phase extraction (SPE) column with 500 μL of methanol and 500 μL of ultrapure water; adding the diluted thick extract to the activated solid phase extraction (SPE) column; allowing it to drain naturally; rinsing the solid phase extraction (SPE) column with 500 μL of a methanol solution with a volume concentration of 30%; discarding the rinsing solution and then eluting with 500 μL of methanol to obtain the eluate.

[0022] According to another aspect of the present invention, the present invention provides a method for determining the content of index components of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction, including: preparing a test sample solution from the pharmaceutical preparation of the test Gualou Xiebai Banxia Decoction; preparing a second mixed reference substance solution from loliolide, chrysoeriol, rutin, cucurbitacin D, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -Timosaponin B II and PWVPG; injecting the test sample solution and the second mixed reference substance solution into a liquid chromatography - mass spectrometry instrument respectively, and obtaining the test spectrum of the test sample solution and the mixed reference spectrum of the second mixed reference substance solution according to the liquid chromatography - mass spectrometry method determined by the above construction method; calculating the contents of the index components loliolide, chrysoeriol, rutin, cucurbitacin D, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -Timosaponin B II and PWVPG in the test sample solution according to the concentration of the second mixed reference substance solution, the peak area of the second mixed reference substance solution in the mixed reference spectrum, and the peak area of the components corresponding to the test sample solution and the second mixed reference substance solution in the test spectrum.

[0023] Preferably, the mass spectrometry conditions of the liquid chromatography - mass spectrometry method include: the ion source is ESI, the ionization mode is positive and negative ion switching mode, from 0 to 10.5 min is the positive ion mode, and from 10.5 to 19 min is the negative ion mode; the backflush nitrogen temperature is 325 °C, the backflush nitrogen flow rate is 10.0 L / min, the nebulizing gas flow rate is 40 psi, and the capillary voltage is 4000 V; the index components are determined in the MRM mode.

[0024] Preferably, the following relationship is satisfied between the concentration of the second mixed reference substance solution and the peak area of the second mixed reference substance solution in the mixed reference spectrum: The first regression equation of loliolide is y = 213366.97x + 8304.54 (r 2 = 0.9992, weight is 1 / x), the linear range is 0.05 - 5 µg / mL, and the detection limit is 0.01 µg / mL (S / N≥3); The second regression equation of chrysoeriol is y = 327983.31x + 14.19 (r 2 = 0.9992, weight is 1 / x), the linear range is 0.0025 - 2.5 µg / mL, and the detection limit is 0.0005 µg / mL (S / N≥3); The third regression equation of rutin is y = 364177.34x - 1139.58 (r 2 = 0.9994), the linear range is 0.0125 - 5 µg / mL, and the detection limit is 0.0025 µg / mL (S / N≥3); The fourth regression equation of cucurbitacin D is y = 40933.10x + 85.76 (r 2 = 0.9999), the linear range is 0.025 - 5 µg / mL, and the detection limit is 0.005 µg / mL (S / N≥3); The fifth regression equation of Macrostemonoside I is y = 87781.47x - 5630.33 (r 2 = 0.9994), the linear range is 0.15 - 15 µg / mL, and the detection limit is 0.0375 µg / mL (S / N≥3); The 25 S -Timosaponin B II's sixth regression equation is y = 286470.28x - 18018.18 (r 2 = 0.9992, weight is 1 / x), the linear range is 0.1 - 10 µg / mL, and the detection limit is 0.025 µg / mL (S / N≥3); The 25 R -Timosaponin B II's seventh regression equation is y = 196461.26x - 12840.04 (r 2 = 0.9994), the linear range is 0.25 - 25 µg / mL, and the detection limit is 0.05 µg / mL (S / N≥3); The eighth regression equation of PWVPG is y = 1185110.80x - 3069.17 (r 2=0.9994, weight is 1 / x), the linear range is 0.005-1 µg / mL, and the detection limit is 0.001 µg / mL (S / N≥3); wherein, in the first regression equation to the eighth regression equation, the abscissa is the concentration of the corresponding component of the second mixed reference solution, and the ordinate is the peak area in the mixed control spectrum.

[0025] Preferably, the indicative ingredients are ryegrass lactone, rutin, cucurbitacin D, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -MRM scan parameters for Timosaponin B II and PWVPG were:

[0026]

[0027] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a method for constructing a fingerprint of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction and determining the content of an index component, which realizes the effective separation of different types of index components in the pharmaceutical preparation of Gualou Xiebai Banxia Decoction by optimizing the liquid phase elution method, and realizes the identification and identification of the chromatographic peaks in the fingerprint by using the ion and fragment information listed by the mass spectrometer, and at the same time establishes a quantitative analysis method for the index components in the pharmaceutical preparation of Gualou Xiebai Banxia Decoction. The present invention stably and comprehensively improves the evaluation standard of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction, ensures the effectiveness of the clinical use of medicinal materials, and provides a reference for the overall control of the quality of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1 The fingerprints of 15 batches of Gualou Xiebai Banxia decoction in positive ion mode are shown, where the sample numbers are shown in Table 1, and R is the control fingerprint;

[0030] Figure 2 The fingerprints of 15 batches of Gualou Xiebai Banxia decoction in negative ion mode are shown, where the sample numbers are shown in Table 1, and R is the control fingerprint;

[0031] Figure 3 The 12 common peaks in the fingerprint of Gualou Xiebai Banxia decoction in positive ion mode are shown;

[0032] Figure 4Eleven common peaks in the fingerprint of Gualou Xiebai Banxia Decoction in negative ion mode are shown;

[0033] Figure 5 The mixed reference map and the fingerprint of Gualou Xiebai Banxia Decoction in positive ion mode are shown (where peak 2 is PWVPG; peak 3 is AVGTNHLLSGETLD; peak 4 is loline; peak 5 is Macrostemonoside I; peak 8 is 25 S -Timosaponin B II; peak 9 is 25 R -Timosaponin B II);

[0034] Figure 6 The mixed reference substance map and the fingerprint of Gualou Xiebai Banxia Decoction in negative ion mode are shown (where peak 2 is Macrostemonoside I; peak 6 is 25 S -Timosaponin B II; peak 7 is 25 R -Timosaponin B II; peak 11 is chrysoeriol);

[0035] Figure 7 The fingerprint of single herb Gualou, the fingerprint of Gualou -deficient prescription and the fingerprint of the compound prescription in positive ion mode are shown (where peak 4 is loline; peak 10 is Khekadaengenin I);

[0036] Figure 8 The fingerprint of single herb Allium macrostemon, the fingerprint of Allium macrostemon -deficient prescription and the fingerprint of the compound prescription in positive ion mode are shown (where peak 1 is Macrostemonoside G; peak 5 is Macrostemonoside I; peak 8 is 25 S -Timosaponin B II; peak 9 is 25 R -Timosaponin B II; peak 12 is the isomer of Timosaponin B II);

[0037] Figure 9 The fingerprint of single herb Pinellia ternata, the fingerprint of Pinellia ternata -deficient prescription and the fingerprint of the compound prescription in positive ion mode are shown (where peak 2 is PWVPG; peak 3 is AVGTNHLLSGETLD; peak 6 is N -(1-hydroxy-3-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro -2 H -pyran-2-yl)oxy)propan-2-yl)nonadeca-5,8,11,14-tetraenamide; peak 7 is the isomer of peak 6);

[0038] Figure 10 Shows the single-herb spectrum of yellow rice wine, the spectrum of yellow rice wine without certain herbs, and the compound spectrum in the positive ion mode (where peak 11 is a tetrapeptide analog (Pro, Val, N -CH 3 -Val, N -(CH 3 ) 2 -Val));

[0039] Figure 11 Shows the single-herb spectrum of Trichosanthes kirilowii Maxim., the spectrum of Trichosanthes kirilowii Maxim. without certain herbs, and the compound spectrum in the negative ion mode (where peak 1 is 4'-dihydrophaseic acid; peak 4 is Arvenin Ⅲ or its isomer; peak 8 is 23,24-dihydrokhekadaengoside E; peak 9 is Arvenin Ⅲ or its isomer; peak 10 is 25- O -Acetylbryoamaride; peak 11 is chrysoeriol);

[0040] Figure 12 Shows the single-herb spectrum of Allium macrostemon Bunge, the spectrum of Allium macrostemon Bunge without certain herbs, and the compound spectrum in the negative ion mode (where peak 2 is Macrostemonoside I; peak 6 is 25 S -Timosaponin B II; peak 7 is 25 R -Timosaponin B II);

[0041] Figure 13 Shows the single-herb spectrum of yellow rice wine, the spectrum of yellow rice wine without certain herbs, and the compound spectrum in the negative ion mode (where peak 3 is 1,2-Diethyl 3,6-dihydro-3,3,5-trimethyl-1,2

[0042] -pyridazinedicarboxylate; peak 5 is an unidentified compound); and

[0043] Figure 14 Shows the mixed control spectrum and the fingerprint spectrum of Gualou Xiebai Banxia Decoction under the MRM quantitative method. Detailed implementation manners

[0044] The following listed examples are to enable those skilled in the art to understand the present invention more clearly. It should be noted that the following examples do not limit the scope of protection required by the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples can be obtained from commercial channels or by known existing methods without special instructions.

[0045] The information of the drugs used in the formula of the present invention is shown in Table 1. It should be noted that the method for constructing the fingerprint of Gualou Xiebai Banxia Decoction provided by the present invention is applicable to the decoction, paste, dry powder, etc. of Gualou Xiebai Banxia Decoction.

[0046] Table 1: The combination of the prepared pieces of the formula of 15 batches of Gualou Xiebai Banxia Decoction and their corresponding producing areas, and the combination of related single herbs and negative prepared pieces

[0047]

[0048] (Continued from the above table)

[0049] 。

[0050] Example 1

[0051] Method for constructing the fingerprint of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction

[0052] This example provides a method for constructing the fingerprint of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction, including: preparing the pharmaceutical preparation of Gualou Xiebai Banxia Decoction into a test solution; preparing rye-grass lactone, chrysoeriol, 25 S -Timosaponin B Ⅱ, Macrostemonoside I, 25 R -Timosaponin B II, PWVPG and AVGTNHLLSGETLD into a first mixed reference substance solution; injecting the test solution and the first mixed reference substance solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining the fingerprint of the pharmaceutical preparation according to the liquid chromatography-mass spectrometry method. The following provides the specific construction method.

[0053] 1 Optimization of the sample pretreatment method for the pharmaceutical preparation of Gualou Xiebai Banxia Decoction

[0054] 1.1 Preparation of the pharmaceutical preparation of Gualou Xiebai Banxia Decoction

[0055] Take 60.00 g of Trichosanthes kirilowii Maxim., 41.40 g of Allium macrostemon Bge., and 34.50 g of Pinellia ternata (Thunb.) Breit., soak them in 2000 mL of yellow rice wine for 30 minutes, heat and decoct them to 800 mL at 600 W, filter the decoction with a No. 9 sieve (200-mesh sieve) of the Chinese Pharmacopoeia to obtain the decoction liquid, and concentrate the decoction liquid under reduced pressure to 175 mL of thick extract.

[0056] 1.2 Selection of the sample pretreatment method

[0057] Examine different pretreatment methods, including:

[0058] ① Thick extract dilution method: Take an appropriate amount of thick extract, dilute it 1:10 with water as the solvent, dry it by nitrogen blowing, redissolve it with 70% methanol, centrifuge, and take the supernatant for injection and detection;

[0059] ② Diluted thick extract - extraction method: Take an appropriate amount of thick extract, dilute it 1:10 with water as the solvent, extract the diluted solution with water-saturated n-butanol three times, combine the extraction solutions, spin-dry them by rotary evaporation, and then redissolve them with 70% methanol, centrifuge, and take the supernatant for injection and detection;

[0060] ③ Diluted thick extract - HLB elution method: Take an appropriate amount of thick extract, dilute it 1:10 with water as the solvent, perform HLB elution on the diluted solution, elute with 5% MeOH to remove macromolecular compounds, and then use MeOH as the elution solvent, elute three times in total, 0.5 mL each time. After drying the MeOH elution solution by nitrogen blowing, redissolve it with 70% methanol, centrifuge, and take the supernatant for injection and detection;

[0061] ④ Diluted thick extract - HLB gradient dilution method: Take an appropriate amount of thick extract, dilute it 1:10 with water as the solvent, perform HLB gradient elution on the diluted solution, elute with 5%, 20%, 50%, and 100% MeOH gradients, elute three times for each gradient, 0.5 mL each time. Collect the elution sample solutions of each gradient, dry them, redissolve them with 70% methanol, centrifuge, and take the supernatant for injection and detection;

[0062] ⑤ Diluted extract - water extraction and alcohol precipitation method: Take an appropriate amount of thick extract, dilute it 1:10 with water as the solvent, add three volumes of ethanol to precipitate macromolecular compounds, let it stand at 4°C for 12 h, take the supernatant, dry it by nitrogen blowing, redissolve it with 70% methanol, centrifuge, and take the supernatant for injection and detection;

[0063] ⑥ Diluted extract - water extraction, alcohol precipitation - extraction method: Take an appropriate amount of thick extract, dilute it 1:10 with water as the solvent, add three volumes of ethanol to precipitate macromolecular compounds, let it stand at 4°C for 12 h, extract the supernatant with water-saturated n-butanol three times, combine the extraction solutions, spin-dry them by rotary evaporation, and then redissolve them with 70% methanol, centrifuge, and take the supernatant for injection and detection.

[0064] The results showed that: ① In the diluted thick extract method and the diluted thick extract - water extraction and alcohol precipitation method, the components of yellow rice wine seriously interfered with the detection of other medicinal flavor components; ② The diluted thick extract - extraction method and the diluted thick extract - water extraction, alcohol precipitation - extraction method would cause the loss of characteristic components in the medicinal materials; By comparing the experimental results of the diluted thick extract - HLB elution method and the diluted thick extract - HLB gradient elution method, it was found that a low-proportion methanol solution (such as elution with 20% MeOH) could remove most of the large-polarity compounds and improve the background interference. Therefore, SPE column gradient elution was selected as the preferred pretreatment method for further optimization, and a suitable impurity removal gradient was selected in the 20% - 50% MeOH gradient.

[0065] 1.3 Determination of the elution gradient of the SPE column

[0066] By comparing the impurity removal capabilities of 25%, 30%, and 35% MeOH, it was found that elution with 30% MeOH could significantly improve the background interference of yellow rice wine, effectively remove large-polarity compounds, and without loss of characteristic components in the medicinal materials. Therefore, 30% MeOH was selected as the optimal impurity removal concentration; at the same time, in order to effectively elute low-polarity characteristic components, after impurity removal with 30% MeOH, the sample was eluted with 100% MeOH and collected.

[0067] 1.4 Determination of sample pretreatment method

[0068] Using an Oasis HLB 3 ccVac Cartridge small column as the SPE elution column, the SPE column elution method was as follows: Take an appropriate amount of the thick extract of Gualou Xiebai Banxia Decoction, dilute it 1:10 with water as the solvent, and take 100 μL of the diluted sample for SPE column elution. The SPE column was activated successively with 500 μL of methanol and 500 μL of ultrapure water for standby; the diluted sample was added to the SPE column, and after natural drainage, 500 μL of a 30% (v / v) methanol solution was added for elution, and the eluate was discarded. Then, 500 μL of methanol was used for elution and collection. The collected eluate was vortex-mixed, dried, dissolved in a certain volume of solution, and waited for injection analysis.

[0069] 2 Preparation of test solution

[0070] After the pretreatment of Gualou Xiebai Banxia Decoction by SPE column elution, the methanol eluate was collected, dried by nitrogen blowing, and redissolved with a 70% methanol solution to obtain the test solution.

[0071] 2.1 Preparation of the first mixed reference solution

[0072] Take appropriate amounts of reference substances of loline, chrysoeriol, 25 S -Timosaponin B II, Macrostemonoside I, 25 R -Timosaponin B II, PWVPG, and AVGTNHLLSGETLD, add a 50% (v / v) methanol solution to prepare a 1 mg / mL mother liquor, and mix to prepare a solution with an appropriate concentration, that is, the first mixed reference solution.

[0073] 2.2 Chromatographic conditions

[0074] Using a CORTECS UPLC T3 Column column (2.1×100 mm, 1.6 μm) as the analytical column, an aqueous solution containing 0.1% (v / v) formic acid was used as mobile phase A, and an acetonitrile solution containing 0.1% (v / v) formic acid was used as mobile phase B. Gradient elution was carried out according to Table 2, the flow rate was 0.3 mL / min, and the column temperature was 30 °C.

[0075] Table 2: Gradient Elution Table of LC-MS Fingerprint

[0076]

[0077] 2.3 Determination of Samples from Different Batches and Identification of Common Peaks

[0078] 2.3.1 Sample Determination

[0079] Take the pharmaceutical preparations of Gualou Xiebai Banxia Decoction prepared from 15 batches of formula decoctions with different batch numbers. According to the established preparation method of the test solution, prepare the test solution for LC-MS fingerprint. Under the specified chromatographic conditions, measure the LC-MS spectra of 15 batches of Gualou Xiebai Banxia Decoction and conduct analysis. Figure 1 The fingerprint of 15 batches of Gualou Xiebai Banxia Decoction provided by the present invention in the positive ion mode Figure 2 The fingerprint of 15 batches of Gualou Xiebai Banxia Decoction provided by the present invention in the negative ion mode, where the sample numbers are shown in Table 1, R is the reference fingerprint, and the peak corresponding to the 25 S -Timosaponin B II reference peak is the S peak.

[0080] 2.3.2 Investigation of Fingerprint Construction Method

[0081] The present invention compared different data processing methods such as total ion current mass chromatogram (TIC), base peak ion current mass chromatogram (BPC), or BPC diagram in the extracted ion chromatogram (ESI) mode for fingerprint analysis and determination of common peaks. Due to the complex components of the Gualou Xiebai Banxia Decoction compound, the total ion current mass chromatogram (TIC) cannot deduct the interference of matrix ions and other impurity peaks, while the base peak ion current mass chromatogram (BPC) also extracts other components except for the common peaks, resulting in a significantly lower signal-to-noise ratio (S / N) of each chromatographic peak compared to the BPC diagram in the extracted ion chromatogram (ESI) mode. Through comprehensive comparison, the BPC diagram in the ESI mode is most suitable for fingerprint research.

[0082] 2.3.3 Determination of Common Peaks

[0083] Figure 3 Are 12 common peaks in the fingerprint of Gualou Xiebai Banxia Decoction in the positive ion mode. The results show that a total of 12 characteristic common peaks are detected in the LC-MS fingerprint in the positive ion mode: peaks 1, 5, 8, 9, 12 come from Allium macrostemon Bunge, peaks 2, 3, 6, 7 come from Rhizoma Pinelliae Praeparatum, peaks 4, 10 come from Trichosanthes kirilowii Maxim., and peak 11 comes from yellow rice wine.

[0084] The second peak detected in the LC-MS fingerprint in positive ion mode is the pentapeptide PWVPG, the third peak is the tetradecapeptide AVGTNHLLSGETLD, the fourth peak is loline, the fifth peak is Macrostemonoside I, and the eighth peak is 25 S -Timosaponin B II, and the ninth peak is 25 R -Timosaponin B II, as shown in the following table.

[0085] Table 3: Common peaks detected in the LC-MS fingerprint in positive ion mode and identification

[0086]

[0087] In the LC-MS fingerprint in positive ion mode, taking peak 8 (25 S -Timosaponin B II) as the S peak, the relative retention time ranges of peaks 1-7 and peaks 9-12 relative to the S peak are: 0.328-0.334, 0.367-0.372, 0.406-0.413, 0.494-0.499, 0.545-0.552, 0.667-0.673, 0.738-0.744, 1.034-1.036, 1.221-1.226, 1.637-1.649, 1.787-1.804. Taking peak 8 as the S peak, the RSD values of the relative retention times of peaks 1-7 and peaks 9-12 relative to the S peak are: 0.43%, 0.44%, 0.63%, 0.29%, 0.33%, 0.23%, 0.27%, 0.06%, 0.10%, 0.20%, 0.26%.

[0088] Figure 4 Eleven common peaks in the fingerprint of Gualou Xiebai Banxia Decoction in negative ion mode are shown. A total of 11 characteristic common peaks were detected in the LC-MS fingerprint in negative ion mode: peaks 1, 4, 8, 9, 10, 11 are from Trichosanthes kirilowii Maxim., peaks 2, 6, 7 are from Allium macrostemon Bunge, and peaks 3, 5 are from yellow rice wine, with peak 6 as the reference peak.

[0089] The second peak detected in the LC-MS fingerprint in negative ion mode is Macrostemonoside I, the sixth peak is 25 S -Timosaponin B II, the seventh peak is 25 R -Timosaponin B II, and the eleventh peak is chrysoeriol, as shown in Table 4.

[0090] Table 4: Common peaks detected in the LC-MS fingerprint in negative ion mode and identification

[0091]

[0092] The LC-MS fingerprint in the negative ion mode uses peak 6 (25 S -Timosaponin B II) as the S peak. The relative retention time ranges of peaks 1-5 and peaks 7-11 relative to the S peak are respectively: 0.250 - 0.254, 0.555 - 0.561, 0.699 - 0.729, 0.723 - 0.729, 0.844 - 0.850, 1.035 - 1.039, 1.194 - 1.199, 1.248 - 1.255, 1.613 - 1.622, 1.803 - 1.817. Using peak 6 as the S peak, the RSD values of the relative retention times of peaks 1-5 and peaks 7-11 relative to the S peak are respectively: 0.47%, 0.27%, 0.21%, 0.21%, 0.19%, 0.09%, 0.12%, 0.16%, 0.16% and 0.22%.

[0093] 2.4 Fingerprint Similarity Evaluation

[0094] Figure 5 Shows the mixed control map and the fingerprint of Gualou Xiebai Banxia Decoction in the positive ion mode (where peak 2 is PWVPG; peak 3 is AVGTNHLLSGETLD; peak 4 is loline; peak 5 is Macrostemonoside I; peak 8 is 25 S -Timosaponin B II; peak 9 is 25 R -Timosaponin B II); Figure 6 Shows the mixed control map and the fingerprint of Gualou Xiebai Banxia Decoction in the negative ion mode (where peak 2 is Macrostemonoside I; peak 6 is 25 S -Timosaponin BII; peak 7 is 25 R -Timosaponin B II; peak 11 is chrysoeriol).

[0095] Import the LC-MS fingerprint txt format of 15 batches of test solution of Gualou Xiebai Banxia Decoction into the software "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version), and calculate the similarity of each fingerprint. The results are shown in Table 5 and Table 6.

[0096] Table 5: Similarity of LC-MS Fingerprints of 15 Batches of Gualou Xiebai Banxia Decoction in the Positive Ion Mode

[0097]

[0098] Table 6: Similarity of LC-MS Fingerprints of 15 Batches of Gualou Xiebai Banxia Decoction in the Negative Ion Mode

[0099]

[0100] The results showed that in the positive ion mode, with the chromatographic peak of 25 S -Timosaponin B Ⅱ (peak 8) as the reference peak S, a control chromatogram was generated by the average method, and a control fingerprint of Gualou Xiebai Banxia Decoction was established. The superimposed fingerprint chromatogram of Gualou Xiebai Banxia Decoction was obtained, and the similarity was calculated. The similarities of the fingerprint chromatograms of 15 batches of Gualou Xiebai Banxia Decoction were between 0.930 and 0.984, and the similarities were all higher than 0.9. In the negative ion mode, with the chromatographic peak of 25 S -Timosaponin B Ⅱ (peak 6) as the reference peak S, a control chromatogram was generated by the average method, and a control fingerprint of Gualou Xiebai Banxia Decoction was established. The superimposed fingerprint chromatogram of Gualou Xiebai Banxia Decoction was obtained, and the similarity was calculated. The similarities of the fingerprint chromatograms of 15 batches of Gualou Xiebai Banxia Decoction were between 0.907 and 0.992, and the similarities were all higher than 0.9.

[0101] 2.5 Chemical Constituent Attribution and Identification of LC-MS Fingerprint

[0102] Using the LC-MS method, in the positive ion mode, by comparing the chromatograms of single herbs, corresponding herbs-deficient chromatograms and compound prescriptions ( Figure 7 , Figure 8 , Figure 9 , Figure 10 ), the attribution of 12 common peaks was determined as follows: peaks 4 and 10 were characteristic components of Trichosanthes kirilowii Maxim.; peaks 1, 5, 8, 9, and 12 were characteristic components of Allium macrostemon Bunge; peaks 2, 3, 6, and 7 were characteristic components of Pinellia ternata (Thunb.) Breit. var. typica Nakai; peak 11 was a characteristic component of yellow rice wine.

[0103] Using the LC-MS method, in the positive ion mode, the first mixed reference substance solution and the test solution were detected. By comparing the retention time of the chromatographic peak of the compound, the exact molecular weight and the fragmentation rule of the secondary chromatogram, a total of 6 chromatographic peaks corresponding to the compounds were determined, which were pentapeptide PWVPG, tetradecapeptide AVGTNHLLSGETLD, loline, Macrostemonoside I, 25 S -Timosaponin B II and 25 R -Timosaponin B II. Using the LC-MS method, by comparing with the reported structures in the literature, the retention time and combining the fragmentation rule of the secondary chromatogram, 6 chromatographic peaks corresponding to the compounds were preliminarily identified, which were Macrostemonoside G or its isomers, N-(1-hydroxy-3-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2 H -pyran-2-yl)oxy)propan-2-yl)nonadeca-5,8,11,14-tetraenamide or its isomers, Khekadaengenin I or its isomers, Pro-Val-( N -CH 3 -)Val-( N -(CH 3 ) 2 )-Val tetrapeptide analog and Timosaponin B II isomers.

[0104] Using LC-MS method, in the negative ion mode, by comparing the chromatograms of single herbs, corresponding herb-deficient chromatograms and compound prescriptions ( Figure 11 , Figure 12 , Figure 13 ), it was determined that the attribution of 11 common peaks was as follows: peaks 1, 4, 8, 9, 10, and 11 were characteristic components of Trichosanthes kirilowii Maxim.; peaks 2, 6, and 7 were characteristic components of Allium macrostemon Bunge; peaks 3 and 5 were characteristic components of yellow rice wine.

[0105] Using LC-MS method, in the negative ion mode, the first mixed reference solution and the test solution were detected. By comparing the retention time of the chromatographic peaks of the compounds, the exact molecular weight and the fragmentation rules of the secondary spectra, a total of 4 compounds corresponding to the chromatographic peaks were determined, namely Macrostemonoside I, 25 S -Timosaponin B II, 25 R -TimosaponinB II and Chrysoeriol. Using LC-MS method, by comparing with the structures reported in the literature, the retention time and combining the fragmentation rules of the secondary mass spectra, a total of 6 compounds corresponding to the chromatographic peaks were preliminarily identified, namely 4’-dihydrophaseic acid or its isomers, 1,2-Diethyl 3,6-dihydro-3,3,5-trimethyl-1,2-pyridazinedicarboxylate or its isomers, Arvenin Ⅲ or its isomers, 23,24-dihydrokhekadaengoside E or its isomers and 25- O -Acetylbryoamaride or its isomers.

[0106] Example 2

[0107] This embodiment provides a method for determining the content of the index components of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction, including: preparing a test sample solution from the pharmaceutical preparation of the test Gualou Xiebai Banxia Decoction; taking neolin, chrysoeriol, rutin, cucurbitacin D, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -Timosaponin B II and PWVPG to prepare a second mixed reference substance solution; injecting the test sample solution and the second mixed reference substance solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining the test chromatogram of the test sample solution and the mixed reference chromatogram of the second mixed reference substance solution according to the liquid chromatography-mass spectrometry method determined in the method construction of Example 1 (for example, including but not limited to the chromatographic conditions in Example 1); calculating the content of the index components neolin, chrysoeriol, rutin, cucurbitacin D, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -Timosaponin B II and PWVPG in the test sample solution according to the concentration of the second mixed reference substance solution, the peak area of the second mixed reference substance solution in the mixed reference chromatogram, and the peak area of the corresponding components in the test sample solution and the second mixed reference substance solution. The following provides a specific determination method.

[0108] 1 MRM detection parameters of reference substances

[0109] Based on 8 reference substances, including the index components neolin, chrysoeriol, rutin and cucurbitacin D of Trichosanthes kirilowii Maxim.; the index components Macrostemonoside I, 25 S -Timosaponin B II and 25 R -Timosaponin BII of Allium macrostemon Bunge; the index component PWVPG of Pinellia ternata (Thunb.) Breit. var. typica Nakai, a method for MRM quantification of 15 batches of samples of Gualou Xiebai Banxia Huangjiu Decoction was established, and their MRM parameters are shown in Table 7.

[0110] Table 7: MRM detection parameters of 8 reference substances

[0111]

[0112] 2 Selection of experimental conditions for the quantification method

[0113] The UHPLC-QQQ-MS conditions were as follows: The chromatographic column was a CORTECS UPLC T3 Column (2.1×100 mm, 1.6 μm), the flow rate was 0.3 mL / min, and the column temperature was 30 °C. The eluents were an aqueous solution containing 0.1% (v / v) formic acid (mobile phase A) and an acetonitrile solution containing 0.1% (v / v) formic acid (mobile phase B). The injection volume was 2 μL, and gradient elution was carried out according to the following procedure. The chromatogram of the test solution of Gualou Xiebai Banxia Decoction is shown in Figure 14 .

[0114] From 0 to 0.5 min, mobile phase A was from 85% → 85%, and mobile phase B was from 15% → 15%;

[0115] From 0.5 to 3.8 min, mobile phase A was from 85% → 81%, and mobile phase B was from 15% → 19%;

[0116] From 3.8 to 4.5 min, mobile phase A was from 81% → 77%, and mobile phase B was from 19% → 23%;

[0117] From 4.5 to 10.5 min, mobile phase A was from 77% → 77%, and mobile phase B was from 23% → 23%;

[0118] From 10.5 to 12 min, mobile phase A was from 77% → 69%, and mobile phase B was from 23% → 31%;

[0119] From 12 to 16 min, mobile phase A was from 69% → 64%, and mobile phase B was from 31% → 36%;

[0120] From 16 to 16.5 min, mobile phase A was from 64% → 5%, and mobile phase B was from 36% → 95%;

[0121] From 16.5 to 18.9 min, mobile phase A was from 5% → 5%, and mobile phase B was from 95% → 95%;

[0122] From 18.9 to 19 min, mobile phase A was from 5% → 85%, and mobile phase B was from 95% → 15%.

[0123] For mass spectrometry detection, ESI positive and negative ion mode switching was used. It was in the positive ion mode from 0 to 10.5 min and in the negative ion mode from 10.5 to 19 min; the backflush nitrogen temperature was 325 °C, the backflush nitrogen flow rate was 10.0 L / min, the nebulizing gas flow rate was 40 psig, and the capillary voltage was 4000 V. LC-QQQ / MS was used to determine the analyte in the MRM mode.

[0124] 3 Methodology Validation

[0125] The items for methodology validation included detection limit, linear range, precision, accuracy, recovery rate, matrix effect, and stability.

[0126] 3.1 Detection limit and linear range

[0127] Preparation of the second mixed reference substance solution: Weigh precisely 1 mg each of loline, chrysoeriol, rutin, cucurbitacin D, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -Timosaponin B II and PWVPG, add them to a methanol solution with a volume concentration of 50% to prepare a 1 mg / mL mixed reference substance stock solution. Then, take an appropriate amount of the mixed reference substance stock solution and mix it to prepare a solution containing 10 µg of loline, 5 µg of chrysoeriol, 5 µg of rutin, 5 µg of cucurbitacin D, 15 µg of Macrostemonoside I, 25 S -Timosaponin B II 10 µg, 25 R -Timosaponin B II 50 µg, and 2 µg of PWVPG in 1 mL, that is, the second mixed reference substance solution is obtained.

[0128] Gradually dilute the second mixed reference substance solution to prepare a series of working solutions. Dilute the mixed reference substance stock solution to low, medium, and high concentrations as the quality control samples of the second mixed reference substance. The low concentration contains 0.25 µg of loline, 0.125 µg of chrysoeriol, 0.125 µg of rutin, 0.125 µg of cucurbitacin D, 0.375 µg of Macrostemonoside I, 25 S -Timosaponin B II 0.25 µg, 25 R -Timosaponin B II 1.25 µg, and 0.05 µg of PWVPG in 1 mL; the medium concentration contains 1 µg of loline, 0.5 µg of chrysoeriol, 0.5 µg of rutin, 0.5 µg of cucurbitacin D, 1.5 µg of Macrostemonoside I, 25 S -Timosaponin B II 1 µg, 25 R -Timosaponin B II 5 µg, and 0.2 µg of PWVPG in 1 mL; the high concentration contains 2.5 µg of loline, 1.25 µg of chrysoeriol, 1.25 µg of rutin, 1.25 µg of cucurbitacin D, 3.75 µg of Macrostemonoside I, 25 S -Timosaponin B II 2.5 µg, 25 R-Timosaponin B II: 12.5 µg, PWVPG: 0.5 µg. The standard curve graphs use concentration as the abscissa and peak area as the ordinate, perform linear regression, and obtain the standard curves: The first regression equation for loline is y = 213366.97x + 8304.54 (r 2 = 0.9992, weight is 1 / x), the linear range is 0.05 - 5 µg / mL, and the detection limit is 0.01 µg / mL (S / N ≥ 3); The second regression equation for chrysoeriol is y = 327983.31x + 14.19 (r 2 = 0.9992, weight is 1 / x), the linear range is 0.0025 - 2.5 µg / mL, and the detection limit is 0.0005 µg / mL (S / N ≥ 3); The third regression equation for rutin is y = 364177.34x - 1139.58 (r 2 = 0.9994), the linear range is 0.0125 - 5 µg / mL, and the detection limit is 0.0025 µg / mL (S / N ≥ 3); The fourth regression equation for cucurbitacin D is y = 40933.10x + 85.76 (r 2 = 0.9999), the linear range is 0.025 - 5 µg / mL, and the detection limit is 0.005 µg / mL (S / N ≥ 3); The fifth regression equation for MacrostemonosideI is y = 87781.47x - 5630.33 (r 2 = 0.9994), the linear range is 0.15 - 15 µg / mL, and the detection limit is 0.0375 µg / mL (S / N ≥ 3); 25 S -Timosaponin B II's sixth regression equation is y = 286470.28x - 18018.18 (r 2 = 0.9992, weight is 1 / x), the linear range is 0.1 - 10 µg / mL, and the detection limit is 0.025 µg / mL (S / N ≥ 3); 25 R -Timosaponin B II's seventh regression equation is y = 196461.26x - 12840.04 (r 2 = 0.9994), the linear range is 0.25 - 25 µg / mL, and the detection limit is 0.05 µg / mL (S / N ≥ 3); PWVPG's eighth regression equation is y = 1185110.80x - 3069.17 (r 2 = 0.9994, weight is 1 / x), the linear range is 0.005 - 1 µg / mL, and the detection limit is 0.001 µg / mL (S / N ≥ 3).

[0129] 3.2 Precision, Accuracy, Recovery Rate, Stability, Matrix Effect

[0130] The low, medium, and high concentration quality control samples were each detected 3 times on the same day, and the relative standard deviation (RSD) values of the peak areas of 8 analytes were calculated to obtain the within-day precision; the low, medium, and high concentration quality control samples were injected and detected 3 times every day for three consecutive days to obtain the between-day precision. Accuracy was evaluated by the percentage ratio of the detected concentration to the actual concentration of the quality control samples. The results showed that the within-day and between-day precisions and accuracies of the low, medium, and high concentration quality control samples were within ±15%, meeting the requirements.

[0131] The recovery experiment and matrix effect experiment were carried out by adding a mixed standard solution with a determined concentration to the sample. The recovery rate and matrix effect were obtained according to the following formulas. The results showed that the recovery rate was between 85% - 115%, and the matrix effect was between 85% - 115%, meeting the requirements.

[0132] Recovery rate = (Peak area of the spiked sample before sample pretreatment - Peak area of the sample) / Peak area of the mixed standard solution with a determined concentration.

[0133] Matrix effect = (Peak area of the spiked sample after sample pretreatment - Peak area of the sample) / Peak area of the mixed standard solution with a determined concentration.

[0134] Stability was calculated from the peak areas of the same low, medium, and high concentration quality control samples injected and detected within 48 h at 4°C. The results showed that the RSD of the stability of the low, medium, and high concentration quality control samples was within ±15%, meeting the requirements.

[0135] The above results (Table 8, Table 9) indicated that the established quantitative method could quantitatively analyze the target components in the Gualou Xiebai Banxia Decoction samples stably and accurately.

[0136] 4 Determination of the Contents of Target Components in 15 Batches of Gualou Xiebai Banxia Decoction Samples

[0137] The sample solution to be tested was prepared according to the method in Example 1, and injected and detected under the UHPLC-QQQ-MS conditions in "2 Selection of Quantitative Method Experimental Conditions" in Example 2. The peak areas were recorded, and the contents of loliolide in the Gualou Xiebai Banxia Decoction samples were calculated to be 0.00047% - 0.00223%, chrysoeriol 0.00011% - 0.00043%, rutin 0.00004% - 0.00113%, cucurbitacin D 0.00008% - 0.00188%, Macrostemonoside I 0.00268% - 0.00718%, S -Timosaponin B II 0.00137% - 0.00397%, R- Timosaponin B II: 0.01149% - 0.03693%, PWVPG: 0.00017% - 0.00196%. The specific measurement results are shown in Table 10.

[0138] Table 8: Precision and Accuracy of Methodology Validation

[0139]

[0140] Table 9: Stability, Spiked Recovery, and Matrix Effect of Methodology Investigation

[0141]

[0142] Table 10: Quantitative Results of Index Components in 15 Batches of Gualou Xiebai Banxia Decoction Samples

[0143]

[0144] * The content calculation formula is: the content in Gualou Xiebai Banxia Decoction samples = m / M × 100% (where m is the content of the compound to be detected in the sample detected by linearity, and M is the theoretical mass of the corresponding attributed medicinal material in the detection solution).

[0145] In summary, the present invention provides a method for constructing a fingerprint of Gualou Xiebai Banxia Decoction based on LC - MS, identifying and determining the characteristic components in four medicinal flavors, and constructing the fingerprint of Gualou Xiebai Banxia Decoction. In the positive and negative ion modes, the similarities of 15 batches of Gualou Xiebai Banxia Decoction samples are between 0.930 - 0.984 and 0.907 - 0.992 respectively. A quantitative detection method is established by the MRM mode in UHPLC - QQQ - MS to determine the contents of index components in Gualou Xiebai Banxia Decoction samples, including the index components of Trichosanthes kirilowii Maxim., i.e., loline, chrysoeriol, rutin, cucurbitacin D, the index components of Allium macrostemon Bunge, i.e., Macrostemonoside I, 25 S - Timosaponin B II, 25 R - Timosaponin B II, the index component of Pinellia ternata (Thunb.) Breit. var. purpurea (Makino) Hsiao & K. C. Hsia, i.e., PWVPG. The results show that the average contents of loline, chrysoeriol, rutin, cucurbitacin D, Macrostemonoside I, 25 S - Timosaponin B II, 25 R- Timosaponin B II 0.02142%, PWVPG 0.00074%. This method improves the evaluation criteria of Gualou Xiebai Banxia Decoction and provides a reference for the overall and comprehensive quality control of Gualou Xiebai Banxia Decoction.

[0146] In addition, in our subsequent research, we adopted the method of multi-component determination by single marker, that is, by determining an easily available, inexpensive and effective component (as an internal reference substance, such as 25 S - Timosaponin B II), so as to realize the simultaneous determination of the contents of multiple components (such as other types of Anemarrhena asphodeloides saponins), and then solve the problem of insufficient reference substances while reducing the detection cost.

[0147] The above are the preferred embodiments of the invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction, characterized in that, it includes: Preparing the pharmaceutical preparation of Gualou Xiebai Banxia Decoction into a test sample solution, and the preparation method of the test sample solution includes: adding yellow rice wine to Trichosanthes kirilowii Maxim., Allium macrostemon Bunge and Pinellia ternata (Thunb.) Breit., decocting and concentrating, and filtering to obtain a decoction; concentrating the decoction under reduced pressure to obtain a thick extract; treating the diluted thick extract with a solid phase extraction SPE column to obtain an eluate; redissolving the dried eluate to a certain concentration to obtain the test sample solution, wherein the solid phase extraction SPE column is a hydrophilic-lipophilic balance column HLB; Take lactucin, chrysoeriol, 25 S -Timosaponin B Ⅱ, Macrostemonoside I, 25 R -Timosaponin B II, pentapeptide PWVPG and tetrapeptide AVGTNHLLSGETLD were prepared into the first mixed reference substance solution; Injecting the test sample solution and the first mixed reference substance solution into a liquid chromatography-mass spectrometry instrument respectively, and obtaining the fingerprint of the pharmaceutical preparation according to the liquid chromatography-mass spectrometry method; The chromatographic conditions of the liquid chromatography-mass spectrometry method include: Stationary phase: a chromatographic column filled with octadecylsilane-bonded silica gel; Mobile phase: mobile phase A is water containing 0.1% (v / v) formic acid, and mobile phase B is acetonitrile solution containing 0.1% (v / v) formic acid; Gradient elution is adopted, and the gradient elution is carried out according to the following procedure: 0 - 0.5 min, mobile phase A from 85% → 85%, mobile phase B from 15% → 15%; 0.5 - 3.8 min, mobile phase A from 85% → 81%, mobile phase B from 15% → 19%; 3.8 - 4.5 min, mobile phase A from 81% → 77%, mobile phase B from 19% → 23%; 4.5 - 10.5 min, mobile phase A from 77% → 77%, mobile phase B from 23% → 23%; 10.5 - 12 min, mobile phase A from 77% → 69%, mobile phase B from 23% → 31%; 12 - 16 min, mobile phase A from 69% → 64%, mobile phase B from 31% → 36%; 16 - 16.5 min, mobile phase A from 64% → 5%, mobile phase B from 36% → 95%; 16.5 - 18.9 min, mobile phase A from 5% → 5%, mobile phase B from 95% → 95%; 18.9 - 19 min, mobile phase A from 5% → 85%, mobile phase B from 95% → 15%; The mass spectrometry conditions of the liquid chromatography-mass spectrometry method include: The ion source is ESI; the backflush nitrogen temperature is 325 °C; the backflush nitrogen flow rate is 11.0 L / min; the nebulizer gas flow rate is 45 psig; the auxiliary nitrogen temperature is 300 °C; the auxiliary nitrogen flow rate is 11.0 L / min; the capillary inlet voltage is 3500 V; the Agilent Jet Stream outlet voltage in positive ion mode is 500 V (+), and in negative ion mode is 1500 V (-); the in-source collision voltage is 175 V; the cone voltage is 65.0 V; the Octopole RF Peak is 750 V; the scan mode is full scan; the scan range is m / z 60 ~ 1700.

2. According to the construction method described in claim 1, characterized in that, In the positive ion mode, the fingerprint contains 12 characteristic peaks, and the numbers of the characteristic peaks are peak 1A - peak 12A; The identification of the characteristic peaks and the characteristic peaks is as follows: 。 3. According to the construction method described in claim 2, characterized in that, In the negative ion mode, the fingerprint contains 11 characteristic peaks, and the numbers of the characteristic peaks are peak 1B - peak 11B; The identification of the characteristic peaks and the characteristic peaks is as follows: 。 4. According to the construction method described in claim 3, characterized in that, Taking peak 8A as the S peak, the relative retention time ranges of peaks 1A to 7A and peaks 9A to 12A relative to the S peak are respectively: 0.328 - 0.334, 0.367 - 0.372, 0.406 - 0.413, 0.494 - 0.499, 0.545 - 0.552, 0.667 - 0.673, 0.738 - 0.744, 1.034 - 1.036, 1.221 - 1.226, 1.637 - 1.649, 1.787 - 1.

804.

5. The construction method according to claim 4, wherein, taking peak 6B as the S peak, the relative retention time ranges of peaks 1B to 5B and peaks 7B to 11B relative to the S peak are respectively: 0.250 - 0.254, 0.555 - 0.561, 0.699 - 0.729, 0.723 - 0.729, 0.844 - 0.850, 1.035 - 1.039, 1.194 - 1.199, 1.248 - 1.255, 1.613 - 1.622, 1.803 - 1.

817.

6. The construction method according to any one of claims 1 to 5, wherein, the preparation method of the test sample solution comprises: adding 2000 mL of yellow rice wine to Trichosanthes kirilowii Maxim., Allium macrostemon Bunge and Pinellia ternata (Thunb.) Breit. var. typica Nakai, decocting and concentrating to 800 mL, and obtaining a decoction after filtration.

7. A method for determining the content of the index components of a pharmaceutical preparation of Gualou Xiebai Banxia Decoction, wherein, it comprises: preparing the pharmaceutical preparation of the test Gualou Xiebai Banxia Decoction into a test sample solution; Take rye grass lactone, chrysoeriol, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -Timosaponin B II and pentapeptide PWVPG are prepared into a second mixed reference substance solution; injecting the test sample solution and the second mixed reference substance solution into a liquid chromatography - mass spectrometry instrument respectively, and obtaining a test spectrum of the test sample solution and a mixed reference spectrum of the second mixed reference substance solution according to the liquid chromatography - mass spectrometry method determined by any one of claims 1 to 5. Calculate the contents of the index components, i.e., lolineolide, chrysoeriol, Macrostemonoside I, 25 S -Timosaponin B II, 25 R -Timosaponin B II and pentapeptide PWVPG in the sample solution to be detected according to the concentration of the second mixed reference solution, the peak area of the second mixed reference solution in the mixed reference spectrum, and the peak area of the components corresponding to the second mixed reference solution in the spectrum of the sample solution to be detected.

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