A method for constructing a characteristic map of a Chinese medicinal product and application thereof

Abstract

This invention belongs to the field of quality testing technology for traditional Chinese medicine preparations, specifically relating to a characteristic chromatogram of Liu Shen Qu (a type of medicinal fermentation) and its pharmaceutical preparations, and further disclosing a method for constructing the characteristic chromatogram and a quality testing method for Liu Shen Qu and its pharmaceutical preparations. The method for constructing the characteristic chromatogram of Liu Shen Qu and its pharmaceutical preparations, using Liu Shen Qu medicinal materials and its pharmaceutical preparations as the detection objects, establishes a characteristic chromatogram method for the pharmaceutical preparations based on ultra-high performance liquid chromatography (UHPLC), and identifies nine common characteristic peaks using traditional Chinese medicine chromatographic fingerprinting. Under these chromatographic conditions, the established characteristic chromatogram shows significant characteristic components and good separation effect, and can simply and quickly identify the types and quantities of chemical components contained in Liu Shen Qu decoction pieces and related preparations (standard decoction freeze-dried powder, formula granules), providing a rapid and reliable detection method for the characteristic chromatogram identification of Liu Shen Qu decoction pieces and related preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality inspection of traditional Chinese medicine preparations, and particularly relates to a characteristic chromatogram of Liushenqu and its pharmaceutical preparations, and further discloses a construction method of the characteristic chromatogram, as well as a quality inspection method for Liushenqu and its pharmaceutical preparations. Background Art

[0002] Liushenqu is a fermented agent made from raw materials such as Polygonum hydropiper, Artemisia annua, Xanthium sibiricum, Phaseolus angularis, Prunus armeniaca, wheat bran, and flour. According to literature reports, its chemical components mainly include amylase, yeast, volatile oil, glycosides, and B vitamins. Liushenqu is a traditional agent, and there is no national pharmacopoeia standard yet. Moreover, due to the complex and diverse chemical components contained in Liushenqu, and the fact that traditional Chinese medicine itself is a system in which complex components work together to exert therapeutic effects, it is impossible to accurately control the quality of medicinal materials in an all-round and multi-range manner. At present, Liushenqu varies in formula, compatibility, fermentation process, etc. in different regions across the country, with large quality differences. The quality control mainly based on morphological identification lacks quantitative indicators, and the quality standard needs to be improved. Also, due to the complexity of the compound composition and natural fermentation, the research on its pharmacodynamic substance basis is relatively weak.

[0003] Liushenqu is made by fermenting multiple traditional Chinese medicines. Many scholars have only studied the fingerprint chromatogram of Liushenqu. For example, the construction method and application of the characteristic chromatogram of Liushenqu pieces, stir-fried Liushenqu pieces and their preparations disclosed in Chinese Patent CN117871722A take Liushenqu pieces, stir-fried Liushenqu pieces, stir-fried Liushenqu standard decoction and related preparations as research objects. Among them, 6 common characteristic peaks were confirmed in Liushenqu pieces, and 3 characteristic peaks, namely peak 1 (uridine), peak 2 (thymine), and peak 6 (guanosine), were identified. By measuring 15 batches of stir-fried Liushenqu pieces prepared from 15 batches of Liushenqu pieces, after Liushenqu pieces were stir-fried, the peak area of peak 3 increased significantly, a new gallic acid characteristic peak appeared, and peak 5 degraded significantly and disappeared completely in some batches. Therefore, the chemical composition of Liushenqu changes significantly before and after stir-frying, providing a rapid and reliable detection method for the identification of Liushenqu pieces and stir-fried Liushenqu-related preparations. However, the overall analysis time of the characteristic chromatogram provided by this method is long (60 minutes), the information shown is not rich enough, its elution gradient only shows the large-polarity components with poor retention on the chromatographic column (0% - 2% organic phase), and finally 6 characteristic peaks were calibrated, with few identified known components and lack of specificity. Therefore, establishing a method that can comprehensively and systematically detect Liushenqu and its pharmaceutical preparations, especially a method for establishing the characteristic chromatogram of Liushenqu and its pharmaceutical preparations, is of great significance for its comprehensive quality inspection and overall quality control. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations, so as to comprehensively reflect the intrinsic quality and medication safety of Liushenqu and its pharmaceutical preparations.

[0005] The second technical problem to be solved by the present invention is to provide a quality testing method for Liushenqu and its pharmaceutical preparations.

[0006] To solve the above-mentioned technical problems, the present invention provides a method for constructing characteristic chromatograms of Liushenqu and its pharmaceutical preparations, which includes the step of performing ultra-high performance liquid chromatography detection on the test solution of Liushenqu and / or Liushenqu pharmaceutical preparations.

[0007] Chromatographic conditions included: using octadecylsilane-bonded silica gel as the stationary phase, methanol as mobile phase A, and 0.1% formic acid as mobile phase B, with gradient elution performed according to the following procedure:

[0008] From 0 to 8 minutes, the ratio of A to B was 0% to 3% and 100% to 97%.

[0009] 8-15 min, A:B ratio changed from 3% to 13%; 97%:B ratio changed from 87%.

[0010] Over 15-30 minutes, the ratio of A to B changed from 13% to 38% and from 87% to 62%.

[0011] Specifically, in the method for constructing the characteristic chromatogram of Liushenqu and its pharmaceutical preparations, the chromatographic conditions in the ultra-high performance liquid chromatography detection step further include: a flow rate of 0.3 ml / min, a column temperature of 30℃, and a detection wavelength of 270 nm.

[0012] Specifically, the method for constructing the characteristic spectrum of the Liushenqu and its pharmaceutical preparations includes the following method for preparing the test sample solution: accurately adding the test sample to the first solvent and mixing, then ultrasonically treating it, taking the supernatant and treating it with macroporous adsorption resin, eluting it with the second solvent, evaporating it to dryness and redissolving it to obtain the final product.

[0013] Specifically, the method for constructing the characteristic spectrum of the Liushenqu and its pharmaceutical preparations further includes the step of preparing a reference solution of the control medicinal material, which specifically includes: taking the Liushenqu reference medicinal material and accurately adding it to the first solvent for mixing, collecting the extract after heating and reflux, filtering, taking the supernatant for macroporous adsorption resin treatment, eluting with the second solvent, evaporating to dryness and redissolving to obtain the final product.

[0014] Specifically, the method for constructing the characteristic chromatogram of the Liushenqu and its pharmaceutical preparations further includes the step of preparing a reference solution, and the step of constructing the characteristic chromatogram of the reference solution based on the ultra-high performance liquid chromatography.

[0015] The reference standard includes tryptophan;

[0016] The preparation method of the reference solution includes: taking the reference standard and adding it to a second solvent and mixing them to obtain the solution.

[0017] Specifically, the method for constructing the characteristic spectrum of the Liushenqu and its pharmaceutical preparations:

[0018] The first solvent includes water; and / or,

[0019] The second solvent comprises a 30%-50% methanol solution; and / or,

[0020] The macroporous adsorption resin includes macroporous adsorption resin D101.

[0021] Specifically, the method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations, wherein the Liushenqu pharmaceutical preparations include Liushenqu formula granules, Liushenqu decoction pieces, or Liushenqu standard decoction.

[0022] The present invention also discloses a characteristic spectrum and / or a control characteristic spectrum of Liushenqu and its pharmaceutical preparation, wherein the characteristic spectrum or control characteristic spectrum of Liushenqu and its pharmaceutical preparation is constructed by the method described above;

[0023] Preferably, the characteristic spectrum has 9 characteristic peaks, with peak 6 as the S peak, and the relative retention times of the other characteristic peaks relative to peak 6 are within ±10% of a specified value. The specified values ​​of each characteristic peak are: peak 1: 0.25, peak 2: 0.47, peak 3: 0.55, peak 4: 0.58, peak 5: 0.83, peak 7: 1.31, peak 8: 1.75, and peak 9: 1.88.

[0024] This invention also discloses the method for constructing the characteristic spectrum of the aforementioned Liushenqu and its pharmaceutical preparations, and / or the application of the characteristic spectrum and / or control characteristic spectrum of the aforementioned Liushenqu and its pharmaceutical preparations in the field of quality testing of Liushenqu and its pharmaceutical preparations.

[0025] The present invention also discloses a quality testing method for Liushenqu and its pharmaceutical preparations, including the steps of constructing the characteristic spectrum and the control characteristic spectrum according to the method, and the step of comparing the characteristic spectrum with the control characteristic spectrum.

[0026] The method for constructing characteristic chromatograms of Liu Shen Qu (a traditional Chinese medicine) and its pharmaceutical preparations described in this invention uses Liu Shen Qu medicinal materials and its pharmaceutical preparations as the detection objects. Based on ultra-high performance liquid chromatography (UHPLC), a characteristic chromatogram method for this pharmaceutical preparation was established. Nine common characteristic peaks were identified using traditional Chinese medicine chromatographic fingerprinting, and eight characteristic components were identified: peak 1 is uridine, peak 2 is adenosine, peak 3 is guanosine, peak 4 is 5-hydroxymethylfurfural, peak 6 is tryptophan, peak 7 is vanillic acid, peak 8 is ferulic acid, and peak 9 is shampoosine. Under these chromatographic conditions, the established characteristic chromatograms show significant characteristic components and good separation effects in the detection of Liu Shen Qu decoction pieces and related preparations. It can also easily and quickly identify the types and quantities of chemical components contained in Liu Shen Qu decoction pieces and related preparations (standard decoction freeze-dried powder, formula granules). The results shown in the chromatograms are easily interpretable, providing a rapid and reliable detection method for the characteristic chromatogram identification of Liu Shen Qu decoction pieces and related preparations.

[0027] The method for constructing the characteristic chromatograms of Liushenqu and its pharmaceutical preparations described in this invention uses ultra-high performance liquid chromatography (UHPLC) to establish characteristic chromatograms of Liushenqu decoction pieces and related preparations, greatly shortening the analysis time (30 minutes). By increasing the proportion of organic phase (0%–38% organic phase) and setting gradient time, and simultaneously purifying the test sample using D101 macroporous adsorption resin, the final established characteristic chromatogram has a stable baseline, significant characteristic components, and good separation effect. Nine common characteristic peaks were identified, and eight characteristic components were identified: peak 1 is uridine, peak 2 is adenosine, peak 3 is guanosine, peak 4 is 5-hydroxymethylfurfural, peak 6 is tryptophan, peak 7 is vanillic acid, peak 8 is ferulic acid, and peak 9 is shampoosine. This greatly enriches the types and quantities of chemical components in Liushenqu decoction pieces and related preparations, making them more specific. The results shown in the chromatogram are easier to interpret, and the interpretation results are more accurate and reliable.

[0028] The method for constructing the characteristic chromatograms of Liushenqu and its pharmaceutical preparations described in this invention establishes a comprehensive and multi-range technical approach. By establishing HPLC characteristic chromatogram inspection items for Liushenqu decoction pieces and its pharmaceutical preparations, the quality of Liushenqu decoction pieces and downstream products can be initially controlled. This provides a more comprehensive, objective, and rapid quality evaluation method for Liushenqu decoction pieces and pharmaceutical preparations, and is of great significance for their comprehensive quality detection and overall quality control. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0030] Figure 1 The characteristic spectrum of the test sample in Example 1;

[0031] Figure 2 Chromatograms of different extraction solvents in Example 2;

[0032] Figure 3 The results are chromatographical results at the three detection wavelengths of 210nm, 260nm, and 322nm in Example 2;

[0033] Figure 4 The chromatographic results at the five detection wavelengths of 260nm, 270nm, 280nm, 300nm and 325nm in Example 2 are shown.

[0034] Figure 5 The chromatograms are those of different extraction solvents and reconstitution solvents optimized in Example 2;

[0035] Figure 6 The chromatograms are those obtained under different column elution solvent optimizations in Example 2.

[0036] Figure 7 Chromatograms of different column packing materials used in Example 2;

[0037] Figure 8 These are chromatograms from different chromatographic columns in Example 3;

[0038] Figure 9 These are chromatograms of different mobile phase systems in Example 3;

[0039] Figure 10 The chromatograms are for different acidic additives in Example 3;

[0040] Figure 11 The chromatograms are for different concentrations of acidic additives in Example 3;

[0041] Figure 12 Chromatograms at different column temperatures in Example 3;

[0042] Figure 13 The chromatograms are for different injection volumes in Example 3;

[0043] Figure 14 The chromatogram of the lyophilized powder of Shenqu in Example 4 is shown below.

[0044] Figure 15 The chromatogram is from the preparation method investigation 1 in Example 5;

[0045] Figure 16 The chromatogram is from the preparation method investigation 2 in Example 5;

[0046] Figure 17 This is the chromatogram under the secondary optimization conditions in Example 5;

[0047] Figure 18 The results of the specificity assessment in Example 6;

[0048] Figure 19 The results of the precision test in Example 6;

[0049] Figure 20 The results are from the repeatability test in Example 6;

[0050] Figure 21 The intermediate precision (personnel) assessment results are from Example 6;

[0051] Figure 22 The results of the stability test in Example 6;

[0052] Figure 23 The results are from the investigation at different flow rates in Example 6;

[0053] Figure 24 The results were obtained at different column temperatures in Example 6.

[0054] Figure 25 The results are from different chromatographic columns in Example 6. Detailed Implementation

[0055] The instruments and equipment involved in the following embodiments of the present invention include:

[0056] Ultra-high performance liquid chromatograph 1: Thermo Vanquish Flex UHPLC system, including a quaternary solvent manager (Vanquish Quaternary Pump F VF-P20-A), an autosampler (Vanquish Split Sampler FT VF-A10-A-02), an original imported column oven (Vanquish Column Compartment H VH-C10-A-02), a DAD detector (Vanquish VF-D40-A), and a chromatography workstation;

[0057] Ultra-high performance liquid chromatograph 2: Waters Acquity H Class UPLC chromatography system, including a quaternary solvent manager, sample manager-FIT, original imported column oven (Vanquish Column Compartment H VH-C10-A-02), and PDA detector;

[0058] Ultra-high performance liquid chromatograph 3: Agilent 1290Ⅱ Infinity ultra-high performance liquid chromatograph system, G7120A quaternary solvent pump, G7129B autosampler, G7116B original imported column oven, G7117A diode array detector.

[0059] Electronic analytical balances: METTLER TOLEDO MS204, MS36S;

[0060] Ultrasonic Cleaner: KQ-500DB model, Kunshan Ultrasonic Instrument Co., Ltd.

[0061] Column 1: Waters ACQUITY UPLC HSS T3, 2.1×100mm, 1.8μm;

[0062] Column 2: Shim-pack Velox C18 (PF-10-122), 2.1 × 100 mm, 1.8 μm;

[0063] Column 3: Eclipse Plus C18 (PF-10-106), 2.1×100mm, 1.8μm.

[0064] The reagent materials involved in the following embodiments of the present invention are shown in Table 1 below.

[0065] Table 1. Reagent and Material Information

[0066]

[0067] In the following embodiments of the present invention, the information on the reference standards involved is shown in Table 2 below.

[0068] Table 2 Information on Reference Standards

[0069]

[0070] The sample information involved in the following embodiments of the present invention is shown in Table 3 below.

[0071] Table 3 Sample Information

[0072]

[0073] Example 1

[0074] The detection method for the characteristic chromatogram of the Liushenqu drug preparation described in this embodiment uses ultra-high performance liquid chromatography (UHPLC) to detect the test sample solution. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel is used as the packing material; the chromatographic column is a Waters ACQUITYUPLC HSS T3, 2.1×100mm, 1.8μm; methanol is used as mobile phase A, and 0.1% formic acid is used as mobile phase B. Gradient elution is performed according to the program shown in Table 4 below, with a flow rate of 0.3ml per minute; the column temperature is 30℃; and the detection wavelength is 270nm.

[0075] Table 4 Gradient elution program

[0076]

[0077] Preparation of the test solution: Accurately weigh approximately 0.5 g of lyophilized Shenqu powder and place it in a stoppered conical flask. Accurately add 5 ml of water, weigh the solution, sonicate to dissolve, centrifuge for 5 minutes, and take the supernatant. Pass the supernatant through a D101 macroporous adsorption resin (1 cm inner diameter, 20 cm column height; wet packing). Elute with 35 ml of 40% methanol, collect the eluent, evaporate to dryness, redissolve in 2 ml of 40% methanol, filter, and take the filtrate.

[0078] Preparation of reference solution: Accurately weigh 2g of Liu Shen Qu (a traditional Chinese medicine) reference material, add 50ml of water, heat under reflux for 30 minutes, filter, concentrate the filtrate to about 5ml, centrifuge for 5 minutes, take the supernatant, pass it through a D101 macroporous adsorption resin (inner diameter 1cm, column height 20cm; wet packing), elute with 35ml of 40% methanol, collect the eluent, evaporate to dryness, redissolve in 2ml of 40% methanol, filter, and take the filtrate as the reference solution of the reference material. Separately, take the amount of tryptophan reference standard, add 40% methanol to prepare a solution containing 20ug per ml, as the reference solution.

[0079] Assay: Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph. Measure the solution to obtain the characteristic chromatogram of the test sample, as shown in the attached figure. Figure 1 As shown.

[0080] Therefore, the chromatogram of the test sample should show 9 characteristic peaks, which should correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 6 should correspond to the retention time of the reference peak. The peak corresponding to the tryptophan reference standard is peak S. The relative retention times of the remaining characteristic peaks and peak S should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.47 (peak 2), 0.55 (peak 3), 0.58 (peak 4), 0.83 (peak 5), 1.31 (peak 7), 1.75 (peak 8), and 1.88 (peak 9).

[0081] The characteristic peaks were confirmed to be as follows: peak 1 is uridine, peak 2 is adenosine, peak 3 is guanosine, peak 4 is 5-hydroxymethylfurfural, peak 6 is tryptophan, peak 7 is vanillic acid, peak 8 is ferulic acid, and peak 9 is shampoosine.

[0082] Example 2

[0083] Liushenqu is a fermentation agent made by mixing Polygonum hydropiper, Artemisia annua, bitter almond, Xanthium sibiricum, red adzuki bean, wheat bran, and flour. Literature primarily reports its nucleoside, phenolic acid, and flavonoid components. This embodiment optimizes the liquid chromatography method based on the scheme in Example 1, selecting batch number 210901Y of the lyophilized Liushenqu powder as the test sample.

[0084] (1) Extraction solvent

[0085] Considering that Liushenqu lyophilized powder is an aqueous extract with a high content of highly polar components such as nucleosides and phenolic acids, this embodiment initially selected a wavelength of 260 nm for condition optimization. This embodiment investigated the extraction efficiency of Liushenqu lyophilized powder under methanol, 80% methanol, 50% methanol, 20% methanol, and aqueous solvent systems. Chromatograms of the methanol, 80% methanol, 50% methanol, 20% methanol, and aqueous systems under the aforementioned determined chromatographic conditions are shown in the attached figures. Figure 2 As shown in (a)-(e).

[0086] The results showed that the peak profiles of water, 20% methanol, and 50% methanol were quite similar. 80% methanol and methanol exhibited a solvent effect, resulting in poor peak shape and smaller peak area in the initial 0-10 min phase. For nucleoside components, 20% methanol and water provided better peak shapes. However, literature reports that Liushenqu also contains ferulic acid, apigenin, and other phenolic acids and flavonoids; therefore, 50% methanol was tentatively chosen as the extraction solvent for now.

[0087] (2) Selection of detection wavelength (batch number: 210901Y)

[0088] Literature on Liu Shen Qu (a traditional Chinese medicine formula) primarily reports its nucleoside, phenolic acid, and flavonoid components. Adenosine, guanosine, and uridine show significant absorption at 260 nm; vanillic acid, rutin, hyperoside, ferulic acid, quercetin, quercetin, and kaempferol show significant absorption at 258 nm; amygdalin and artemisinin show significant absorption at 210 nm; and ferulic acid is detected at 322 nm. Therefore, this example preliminarily investigated three detection wavelengths: 210 nm, 260 nm, and 322 nm. The results are shown in the appendix. Figure 3 (a)-(c).

[0089] The results showed that there were fewer chromatographic peaks at 210 nm and 322 nm, so a wavelength of 260 nm was selected for further investigation.

[0090] Furthermore, according to literature reports, during the fermentation of Liushenqu, the mass fractions of trigonelline, protocatechuic aldehyde, amygdalin, rutin, hyperoside, scopolamine, and astragaloside showed a decreasing trend, while the mass fractions of gallic acid, protocatechuic acid, vanillic acid, luteolin, and apigenin showed an increasing trend. The mass fractions of cryptochlorogenic acid, caffeic acid, ferulic acid, quercetin, quercetin, and isochlorogenic acid B fluctuated. The maximum absorption wavelengths of each component are as follows: adenosine (260nm), guanosine (260nm), vanillic acid (260nm), protocatechuic acid (260nm), schafod (270nm), gallic acid (270nm), tryptophan (280nm), 5-hydroxymethylfurfural (285nm), trans-coumaric acid (300nm), ferulic acid (325nm), cryptochlorogenic acid (325nm), caffeic acid (325nm), isochlorogenic acid B (325nm), apigenin (340nm), luteolin (350nm), quercetin (350nm), hyperoside (360nm), rutin (360nm), kaempferol (375nm), and quercetin (375nm). Therefore, this embodiment further investigated five detection wavelengths: 260nm, 270nm, 280nm, 300nm, and 325nm. The results are shown in the appendix. Figure 4 As shown in (a)-(e).

[0091] The results showed that there were fewer chromatographic peaks at 300 nm and 325 nm. The overall profiles of 260 nm, 270 nm and 280 nm were similar, with only slight differences in the responses of each chromatographic peak. The responses of each chromatographic peak were higher at 270 nm and 280 nm. At the wavelength of 280 nm, the chromatographic peaks at about 17.8 min and about 24.9 min had higher responses than those at 270 nm. Therefore, the wavelength of 280 nm was selected for further investigation.

[0092] (3) Optimization of extraction solvent and reconstitution solvent

[0093] During the aforementioned investigation of detection wavelengths, it was found that the sample peak area was relatively small. Therefore, based on the results of the extraction solvent selection, this embodiment uses 50% methanol as the extraction solvent to increase the sample concentration.

[0094] Preparation method of the test sample: Accurately weigh approximately 0.5 g of lyophilized Shenqu powder and place it in a stoppered conical flask. Accurately add 5 ml of 50% methanol, weigh the sample, and sonicate (565 W, 37 kHz) for 30 minutes. Cool the sample and weigh it again. Make up the weight loss with 50% methanol, filter the sample, and collect the filtrate. Determine the chromatogram of the test sample solution according to the aforementioned chromatographic conditions, see attached figure. Figure 5 As shown in (a).

[0095] The results showed that as the sample concentration increased, the number of small impurity peaks in the chromatogram increased and the baseline became unstable. Therefore, it was considered to increase the polarity of the extraction solvent to reduce impurity peaks.

[0096] This embodiment further investigated the effect of using 70% methanol as the extraction solvent; the chromatogram is attached. Figure 5 As shown in (b).

[0097] The results showed that the number of small impurity peaks in the chromatogram was not reduced compared to 50% methanol with 70% methanol. Therefore, it was considered to further use methanol, which has lower polarity, as both the extraction solvent and the redissolution solvent. Based on the results of the extraction solvent selection, methanol showed low extraction efficiency; therefore, methanol was used as the extraction solvent, and the sample was concentrated. The chromatogram is attached. Figure 5 As shown in (c).

[0098] The results showed that using methanol as the extraction solvent reduced small impurity peaks and improved the baseline in the chromatogram. However, the peak leading edge in the first 10 minutes was likely due to solvent effects. Therefore, 50% methanol was used as the resolvent. The chromatogram is attached. Figure 5 As shown in (d). The results show that using 50% methanol as the resolution solvent can effectively reduce the solvent effect and improve the peak shape of the chromatographic peaks. Therefore, 50% methanol is used as the resolution solvent.

[0099] In summary, this embodiment selected 50% methanol as the resolution solvent, which effectively reduced the solvent effect. When methanol was used as the extraction solvent, compared with 50% methanol and 70% methanol, there were fewer small impurity peaks in the chromatogram and the baseline was more stable, but the extraction efficiency of methanol was lower. To avoid errors caused by inconsistencies in sample concentration and extraction method, the next step is to fix the sample concentration and extraction method, and examine the extraction effects of methanol, 70% methanol, and 50% methanol respectively.

[0100] The preparation method of the test sample in this embodiment is as follows: Take approximately 0.4g of lyophilized Shenqu powder in three portions, accurately weigh them, and place them in stoppered conical flasks. Accurately add 10ml each of methanol, 70% methanol, and 50% methanol, respectively, weigh them, and sonicate (565W power, 37kHz frequency) for 30 minutes. Cool, weigh again, and replenish the lost weight with the appropriate solvent. Filter, take the filtrate, evaporate to dryness, redissolve in 2ml of 50% methanol, filter, and take the filtrate to obtain the test sample. The chromatographic results of extraction with methanol, 70% methanol, and 50% methanol are shown in the appendix. Figure 5 As shown in (e)-(g).

[0101] The results showed that when methanol was used as the extraction solvent, the chromatographic peak area was significantly lower than that of 70% methanol and 50% methanol, indicating that the extraction efficiency was too low. The extraction effects of 70% methanol and 50% methanol were similar, and the chromatographic peak areas were not significantly different. Taking all factors into consideration, 50% methanol was finally selected as the extraction solvent.

[0102] (4) Optimization of column elution solvent (batch number: 210901Y)

[0103] In this embodiment, 50% methanol was used as the extraction solvent. There were many small impurity peaks in the chromatogram. Therefore, we considered adding a column pretreatment method to improve this problem. We first used a neutral alumina column to investigate the effect of different elution solvents on the sample.

[0104] Preparation method of the test sample: Accurately weigh approximately 0.5 g of lyophilized Shenqu powder and place it in a stoppered conical flask. Add 15 ml of 50% methanol and sonicate (565 W power, 37 kHz frequency) for 30 minutes. Filter and collect the filtrate for later use. Weigh 0.5 g of neutral alumina (100-200 mesh) and pack it into a column. Wash with 15 ml of methanol, collect the filtrate, elute with 30 ml of methanol, collect the eluent, evaporate to dryness, redissolve with 2 ml of 50% methanol, filter, and collect the filtrate to obtain the test sample.

[0105] In this embodiment, the chromatograms of the pre-column sample and the methanol-eluted sample are shown in the appendix. Figure 6 As shown in (a)-(b).

[0106] The results showed that when eluting a neutral alumina column with methanol, five main chromatographic peaks at approximately 5.2 min, 12.4 min, 17.7 min, 24.9 min, and 27.2 min were lost. The peak loss was severe, with very small peak areas, and unknown impurities were also eluted. This may be due to an unsuitable elution solvent.

[0107] Therefore, in this embodiment, the column was further eluted again using a methanol solution containing 1% formic acid. The eluent was concentrated and injected to observe whether the five lost main chromatographic peaks could be eluted. The results are shown in the attached figure. Figure 6 As shown in (c).

[0108] The results showed that the methanol solution containing 1% formic acid eluted the chromatographic peak at about 4-7 min, but the later chromatographic peak was still not eluted. This may be because the neutral alumina column packing was not suitable. Therefore, the next step is to investigate different column packings.

[0109] (5) Optimization of column packing

[0110] Nucleoside components are lost more on neutral alumina columns than on acidic alumina columns. Therefore, acidic alumina columns were used for sample pretreatment, and SHIMSEM Styra HLB columns and Waters OASIS® HLB Artridge columns were also tested.

[0111] Preparation method of test sample (alumina column): Accurately weigh approximately 0.5 g of lyophilized Shenqu powder and place it in a stoppered conical flask. Add 15 ml of 50% methanol and sonicate (565 W, 37 kHz) for 30 minutes. Filter and collect the filtrate. Weigh 0.5 g of acidic alumina / neutral alumina (100-200 mesh) and pack it into the column. Wash with 15 ml of methanol containing 1% formic acid. Load the sample onto the filtrate and elute with 30 ml of methanol containing 1% formic acid. Collect the eluent, evaporate to dryness, redissolve in 2 ml of 50% methanol, filter, and collect the filtrate.

[0112] Preparation of the test sample (using an HLB column): Accurately weigh approximately 0.2 g of lyophilized Shenqu powder and place it in a stoppered conical flask. Add 10 ml of 50% methanol and sonicate (565 W, 37 kHz) for 30 minutes. Filter and collect the filtrate. Activate the SHIMSEM Styra HLB column / Waters OASIS® HLB Cartridge by rinsing with 6 ml of methanol, then equilibrate with 4 ml of water. Load the filtrate onto the sample, rinse with 6 ml of an aqueous solution containing 1% methanol, discard the eluent, and then elute with 20 ml of methanol. Collect the eluent, evaporate to dryness, redissolve in 1 ml of 50% methanol, filter, and collect the filtrate.

[0113] In this embodiment, the chromatograms of samples before column chromatography (0.5g-15ml), samples after chromatography on neutral alumina columns (0.5g-2ml), samples after chromatography on acidic alumina columns (0.5g-2ml), samples before column chromatography (0.5g-5ml), samples after chromatography on acidic alumina columns (0.2g-1ml), samples after chromatography on SHIMSEM Styra HLB columns (0.2g-1mL), and samples after chromatography on Waters OASIS® HLB Cartridge columns (0.2g-1mL) are shown in the appendix. Figure 7 As shown in (a)-(g).

[0114] The results showed that neutral alumina column resulted in significant peak loss, with a reduction in the number of peaks and a substantial decrease in peak area. Acidic alumina column retained most of the major chromatographic peaks and removed minor peaks, effectively improving the separation of peaks from 5 to 15 min. However, it lost a peak at approximately 27.2 min, with a peak area loss of about 50%. The number of peaks and the proportion of each peak area were consistent between the two column-treated samples, indicating good reproducibility of the sample pretreatment method. HLB column treatment only removed the peaks from the first 5 minutes and did not improve other peaks. Considering all factors, acidic alumina was ultimately selected as the packing material.

[0115] In summary, 50% methanol and 70% methanol have similar extraction effects, but the sample chromatograms contain many small impurity peaks. Methanol as an extraction solvent can reduce the number of small impurity peaks, but its extraction efficiency is significantly lower than that of 50% methanol and 70% methanol. Therefore, 50% methanol was ultimately chosen as the extraction solvent. Using 50% methanol as a redissolving solvent can effectively reduce the solvent effect and improve the peak shape of the first half of the chromatogram; therefore, 50% methanol was chosen as the redissolving solvent. Among the three different column packing materials—neutral alumina, acidic alumina, and HLB—acidic alumina is superior. The acidic alumina column can retain most of the main chromatographic peaks, effectively improving the separation of the first half of the chromatographic peaks, and has good reproducibility. However, it loses the chromatographic peak at approximately 27.2 min, with a peak area loss of about 50%.

[0116] When the sample concentration is 0.1 mg / mL, the peak area of ​​the main chromatographic peak is approximately 60–334 (mAU*s), which is suitable. Considering the simplicity of operation, the method of preparing the sample by ultrasonic extraction with 50% methanol without concentration (0.5 g–5 mL) was chosen. However, since peralumina columns can effectively reduce small impurity peaks, make the baseline more stable, ensure better separation, and remove complex matrices while protecting the column, the sample preparation method using peralumina columns can be considered as an alternative.

[0117] Example 3

[0118] This embodiment optimizes the chromatographic conditions based on the chromatographic conditions established in Example 1, and selects the batch number of Liushenqu lyophilized powder as the test sample: 210901Y.

[0119] (1) Column optimization

[0120] This embodiment investigated the effect of chromatographic columns from different manufacturers and with different packing materials on the separation effect. Specific column information is shown in Table 5 below, and the results are attached. Figure 8 in (a)-(e).

[0121] Table 5 Information on different chromatographic columns

[0122]

[0123] The results showed that Waters ACQUITY UPLC HSS T3 had better separation of the first half of the chromatographic peaks and better peak shape. Therefore, Waters ACQUITY UPLC HSS T3 was selected for further investigation.

[0124] (2) Optimization of mobile phase system

[0125] According to literature reports, Liushenqu contains nucleosides, phenolic acids, and flavonoids. Considering the high polarity of these components, this experiment initially selected methanol, which has weak elution ability, as the mobile phase to improve the separation of these components. Chromatographic results under different mobile phase systems are attached. Figure 9 As shown in (a)-(b).

[0126] The results showed that when methanol was used as the mobile phase, the chromatographic peak separation was poor in the first 15 minutes, and simply slowing down the gradient did not improve the separation effect. Therefore, acetonitrile was considered as the mobile phase, and the results are shown in the attached figure. Figure 9 As shown in (c).

[0127] The results showed that using acetonitrile as the mobile phase resulted in severe peak encapsulation, and because acetonitrile has a strong eluting ability, the proportion of acetonitrile during elution was very small, leading to a very small range of adjustable gradient. Therefore, methanol was chosen as the mobile phase to focus on separating the first-stage chromatographic peaks.

[0128] (3) Optimization of different acid additives

[0129] Considering the presence of phenolic acids, acidic additives are commonly used in the literature. Previous studies have found that the peak order and resolution of nucleoside components are significantly affected by pH. Therefore, this experiment investigated the effects of three acidic additives: 0.05% phosphoric acid (pH=2.42), 0.1% formic acid (pH=2.74), and 0.1% acetic acid (pH=3.32) on the separation effect. The results are shown in the appendix. Figure 10 As shown in (a)-(c).

[0130] The results showed that the resolution of the chromatographic peaks in the 0-15 min range was significantly affected by pH; both excessively high and low acidity were detrimental to the separation of the initial chromatographic peaks. When 0.1% formic acid (pH=2.74) was used as a mobile phase additive, the peak shapes and resolution of each chromatographic peak in the 0-15 min range were better. Therefore, 0.1% formic acid (pH=2.74) was ultimately selected for further investigation.

[0131] (4) Optimization of acid additive concentration

[0132] The results of the aforementioned investigations using different acidic additives show that the peak resolution of the first stage of Liushenqu (a traditional Chinese medicine) is significantly affected by pH. Therefore, based on the above results, the concentration of formic acid as a mobile phase additive was investigated. The chromatographic results for the 0.05% formic acid (pH=2.90), 0.1% formic acid (pH=2.74), 0.15% formic acid (pH=2.63), and methanol-0.2% formic acid (pH=2.57) systems are attached. Figure 11 As shown in (a)-(d).

[0133] The results showed that the pH values ​​of different concentrations of formic acid did not differ significantly, but had a significant impact on the chromatographic peak at 5-15 min. Compared with the other three concentrations of formic acid, 0.1% formic acid (pH=2.74) had better resolution of the chromatographic peak at 5-15 min, so 0.1% formic acid (pH=2.74) was finally selected.

[0134] (5) Column temperature optimization

[0135] Because the initial peak of the lyophilized chromatogram of Liushenqu (a traditional Chinese medicine) was poorly separated, we attempted to change the column temperature to obtain better resolution. This experiment investigated the effects of different column temperatures (35℃, 30℃, and 25℃) on the separation effect. The results are shown in the appendix. Figure 12 As shown in (a)-(c).

[0136] The results showed that the separation of the chromatographic peaks at 7-13 min and 17-19 min was better when the column temperature was 30℃, so 30℃ was finally selected.

[0137] (6) Optimization of injection volume

[0138] When the injection volume was 2 μL, the chromatographic peak in the first 0-10 min was broad and the chromatographic parameters were poor. Therefore, we attempted to reduce the injection volume to improve the chromatographic peak parameters. This experiment investigated the effects of injection volumes of 1 μL and 2 μL on the chromatographic peaks. The results are shown in the appendix. Figure 13 As shown in (a)-(b).

[0139] The results showed that the peak resolution and tailing factor were better when the injection volume was 1 μL, so the final injection volume of 1 μL was selected.

[0140] Example 4: Chromatographic Peak Identification

[0141] According to literature reports, during the fermentation of Liushenqu (a traditional Chinese medicine), the mass fractions of trigonelline, protocatechuic aldehyde, amygdalin, rutin, hyperoside, scopolamine, and astragaloside showed a decreasing trend, while the mass fractions of gallic acid, protocatechuic acid, vanillic acid, luteolin, and apigenin showed an increasing trend. The mass fractions of cryptochlorogenic acid, caffeic acid, ferulic acid, quercetin, quercetin, and isochlorogenic acid B fluctuated.

[0142] Based on the optimized conditions determined in Example 3 above, the chromatogram of the Liushenqu lyophilized powder (210901) is shown in the attached figure. Figure 14 As shown.

[0143] Mass spectrometry analysis and reference standard identification revealed seven chromatographic peaks: uridine (262 nm), adenosine (257 nm), guanosine (253 nm), 5-hydroxymethylfurfural (283 nm), tryptophan (278 nm), vanillic acid (260 nm), ferulic acid (323 nm), and shampodoside (270 nm). The specific spectra are shown in Table 6. As can be seen, gallic acid (270nm), luteolin (350nm), apigenin (340nm), kaempferol (375nm), hyperoside (360nm), rutin (360nm), quercetin (350nm), quercetin (375nm), amygdalin (210nm), and trans-coumaric acid (300nm) were not found. Cryptochlorogenic acid (325nm), caffeic acid (325nm), isochlorogenic acid B (325nm), protocatechuic acid (260nm), and protocatechuic aldehyde (230nm) had very small peak areas.

[0144] Table 6. Chromatographic peak spectra of the main peaks of the lyophilized Liushenqu sample and the spectra of the reference standard.

[0145]

[0146] Example 5

[0147] This embodiment is a secondary optimization based on the test sample preparation method determined in the previous embodiments.

[0148] Considering that the aforementioned method still has many impurities and an unstable baseline, the sample processing method will continue to be optimized. It is considered that the sample will be eluted with macroporous adsorption resin D101 and elution solvents of different polarities.

[0149] (2) Test sample preparation investigation 1

[0150] Accurately weigh approximately 0.5g of lyophilized Shenqu powder and place it in a stoppered conical flask. Accurately add 5ml of water, weigh, and dissolve by sonication. Centrifuge and collect the supernatant. Pass the supernatant through a D101 macroporous adsorption resin (1cm inner diameter, 20cm column height; wet packing). Elute sequentially with 35ml of 10% methanol, 25% methanol, 50% methanol, and 70% methanol. Collect the eluent fractions, evaporate the filtrate to dryness, and redissolve the residue in 2ml of 50% methanol. Filter the solution to obtain the test solution. Inject the sample under the confirmed chromatographic conditions described above. The specific results are shown in Tables 7-8 below. Chromatograms for the 10% methanol, 25% methanol, 50% methanol, and 70% methanol systems are shown in the appendix. Figure 15 As shown in (a)-(d).

[0151] Table 7 Relative retention times of each characteristic peak

[0152]

[0153] Table 8. Relative peak areas of each characteristic peak

[0154]

[0155] It can be seen that the sample baseline is more stable and impurity peaks are reduced after passing through the D101 macroporous resin packing column. The sample eluted with 10% methanol and then evaporated and reconstituted lacks 5-hydroxymethylfurfural, vanillic acid, ferulic acid and schafod. The sample eluted with 25% methanol and then evaporated and reconstituted lacks schafod. The characteristic peaks of the samples eluted with 50% methanol and 70% methanol are present, and the peak shape of 50% methanol is better. Further refinement of the methanol elution concentration should be considered.

[0156] (2) Test sample preparation investigation 2

[0157] Accurately weigh approximately 0.5g of lyophilized Shenqu powder and place it in a stoppered conical flask. Accurately add 5ml of water, weigh, and dissolve by sonication. Centrifuge and collect the supernatant. Pass the supernatant through a D101 macroporous adsorption resin (1cm inner diameter, 20cm column height; wet packing). Elute sequentially with 35ml of 40% methanol, 50% methanol, and 60% methanol. Collect the eluent fractions, evaporate the filtrate to dryness, and redissolve the residue in 2ml of 50% methanol. Filter the solution to obtain the test solution. Inject the sample under the confirmed chromatographic conditions described above. The specific results are shown in Tables 9-10 below. Chromatograms for the 30%, 40%, 50%, and 60% methanol systems are shown in the appendix. Figure 16 As shown in (a)-(d).

[0158] Table 9. Relative retention times of each characteristic peak

[0159]

[0160] Table 10 Relative Peak Areas of Each Characteristic Peak

[0161]

[0162] As can be seen from the chromatogram, some characteristic peaks were missing when eluted with 30% methanol and 60% methanol. Compared with 50% methanol elution, 40% methanol elution resulted in higher peak response and better peak shape. Therefore, 40% methanol elution was used as the elution solvent for preparing the characteristic chromatogram of Liushenqu lyophilized powder.

[0163] In summary, after two rounds of optimization, the chromatographic method determined in this embodiment is as follows.

[0164] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0502, Chinese Pharmacopoeia 2020 Edition).

[0165] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; methanol was used as mobile phase A and 0.1% formic acid was used as mobile phase B, and gradient elution was performed according to Table 11 below; the flow rate was 0.3 ml per minute; the column temperature was 30℃; and the detection wavelength was 270 nm.

[0166] Table 11 Gradient elution program

[0167]

[0168] Preparation of the test solution: Accurately weigh approximately 0.5 g of lyophilized Shenqu powder and place it in a stoppered conical flask. Accurately add 5 ml of water, weigh the solution, dissolve it by sonication, centrifuge, and pass the supernatant through a column (20 cm macroporous adsorption resin, wet packing). Pass the solution through the column with 35 ml of 40% methanol. After passing through the column, evaporate the solution to dryness, redissolve it with 2 ml of 50% methanol, filter, and collect the filtrate to obtain the test solution.

[0169] Assay: Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph. Measure the solution to obtain the chromatogram (see attached figure). Figure 17 As shown, peak 1: uridine peak 2: adenosine peak 3: guanosine peak 4: 5-hydroxymethylfurfural peak 6: tryptophan peak 7: vanillic acid peak 8: ferulic acid peak 9: shampoosin.

[0170] Example 6

[0171] This embodiment is based on the chromatographic conditions optimized twice in the aforementioned embodiment 5 for methodological investigation.

[0172] (1) Specificity examination

[0173] Prepare the test solution according to the test solution preparation method, and investigate whether the negative sample (40% methanol) will cause interference. Perform UPLC analysis under the determined chromatographic conditions, and record the chromatogram as attached. Figure 18 The negative blank sample did not produce a peak at the position corresponding to the reference peak, indicating no interference from the blank and demonstrating good method specificity.

[0174] (2) Precision

[0175] The same lyophilized powder test solution (210901Y) was injected six times repeatedly. The relative retention time and relative peak area of ​​the common peaks were measured. The RSD of the relative retention time of each characteristic peak and the reference S peak (peak 6) was less than 2%, and the RSD of the relative peak area of ​​each characteristic peak and the reference S peak (peak 6) was less than 5%, indicating that the instrument has good precision. Detailed results are shown in Tables 12-13 and Appendix. Figure 19 .

[0176] Table 12 Results of Instrument Precision Relative Peak Area Test

[0177]

[0178] Table 13 Results of Instrument Precision Relative Peak Area Test

[0179]

[0180] (3) Method repeatability

[0181] Six samples (210901Y) from the same batch were taken, and the relative retention times and relative peak areas of the common peaks were determined using the characteristic spectral method. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 2.0%, and the RSD values ​​of the relative peak areas of each characteristic peak were also small, indicating that the method has good repeatability. Detailed results are shown in Tables 14-15 and Appendix. Figure 20 .

[0182] Table 14 Results of the method repeatability relative retention time test

[0183]

[0184] Table 15 Results of the method repeatability relative peak area test

[0185]

[0186] (4) Intermediate precision (different operators)

[0187] Three inspectors, at different times, took the same sample of lyophilized Shenqu powder (210901Y), prepared the sample according to the test sample preparation method of the characteristic chromatogram, and used the same equipment to determine the relative retention time and relative peak area of ​​each common peak. The results showed that the RSD values ​​of the relative retention times of all characteristic peaks were less than 2.0%, while the RSD values ​​of the relative peak areas of characteristic peaks 4 and 8 were relatively large, indicating that there were certain differences in the test results among different personnel. Detailed results are shown in Tables 16-17 and the appendix. Figure 21 .

[0188] Table 16 Intermediate Precision Relative Retention Time Test Results (Different Operators)

[0189]

[0190] Table 17 Results of intermediate precision relative peak area tests (by different operators)

[0191]

[0192] (5) Stability test

[0193] Samples from the same batch were prepared and administered according to the characteristic chromatogram method. Injections were performed at 0, 2, 4, 8, 12, and 24 hours, and the relative retention times and relative peak areas of common peaks were measured. The RSD of the relative retention times of each characteristic peak and the reference peak S was less than 2.0%. The RSD of the relative peak area of ​​characteristic peak 4 and the reference peak S was relatively large, but the difference in relative peak area was not significant. These results indicate that the sample solution was stable within 24 hours and met the measurement requirements. Detailed results are shown in Tables 18-19 and the appendix. Figure 22 .

[0194] Table 18 Results of the relative retention time test for stability

[0195]

[0196] Table 19 Results of Stability Relative Peak Area Test

[0197]

[0198] (6) Durability test at different flow rates

[0199] The same sample solution was analyzed using different flow rates according to the characteristic chromatogram determination method. The relative retention time and relative peak area of ​​each characteristic peak were measured. The results showed that the RSD values ​​of the relative retention times of each characteristic peak and the reference peak S were relatively small, while the RSD values ​​of the relative peak areas of peaks 4 and 8 were relatively large, indicating that the flow rate had a certain influence on the method. Therefore, the flow rate was fixed at 0.3 ml / min for the determination. Detailed results are shown in Tables 20-21 below. Chromatograms for systems at 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min are shown in the appendix. Figure 23 (a)-(c).

[0200] Table 20 Results of relative retention time for different flow velocities

[0201]

[0202] Table 21 Results of relative peak area at different flow velocities

[0203]

[0204] (7) Durability test at different column temperatures

[0205] Take the same sample solution and, according to the characteristic chromatogram determination method, analyze the same sample solution at different column temperatures, determining the relative retention time and relative peak area of ​​each characteristic peak. The results are detailed in Tables 22-23 below. Chromatograms at 28℃, 30℃, and 32℃ are shown in the appendix. Figure 24 As shown in (a)-(c).

[0206] Table 22 Results of relative retention times at different column temperatures

[0207]

[0208] Table 23 Results of relative peak area at different column temperatures

[0209]

[0210] The results showed that peak 4 was missing at 28℃, and the relative retention times (RSD) of the other characteristic peaks and the reference peak S were all small, while the relative peak areas (RSD) of some peaks were large. This indicates that column temperature has a certain influence on the method. Therefore, the characteristic spectrum of Liushenqu was determined by fixing the column temperature at 30℃.

[0211] (8) Durability test of different chromatographic columns

[0212] Take the same sample solution and perform characteristic chromatographic determination using different columns according to the method. Column 1: Waters ACQUITY UPLC HSS T3, 2.1×100mm, 1.8μm; Column 2: Shim-pack Velox C18 (PF-10-122), 2.1×100mm, 1.8μm; Column 3: Eclipse Plus C18 (PF-10-106), 2.1×100mm, 1.8μm. Analyze the same sample solution and determine the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 24-25 below. The chromatograms under the conditions of column 1, column 2, and column 3 are shown in the appendix. Figure 25 As shown in (a)-(c).

[0213] Table 24 Relative retention times for different chromatographic columns

[0214]

[0215] Table 25 Relative retention times for different chromatographic columns

[0216]

[0217] The results showed that peaks 4, 5, and 7 were missing on column 2. The relative retention times and relative peak areas of each characteristic peak and the reference peak S were all relatively large, indicating that the chromatographic column had a certain impact on the method. It is recommended to fix the chromatographic column to Waters ACQUITY UPLC HSS T3, 2.1×100mm, 1.8μm for the determination.

[0218] In summary, based on the above methodological investigation results, the nine common peaks in the established Liu Shen Qu standard decoction [characteristic chromatogram] showed little change under various chromatographic conditions. The relative retention times were within ±10%. To improve its robustness, it is recommended that the specified value range be controlled within ±10%.

[0219] Example 7

[0220] This embodiment uses the characteristic spectrum determination of Liu Shen Qu (a traditional Chinese medicine) slices.

[0221] The characteristic chromatograms of multiple batches of Liushenqu (a type of medicinal fermented vegetable) tablets were determined according to the established method for determining the characteristic chromatograms of the standard decoction of Liushenqu. Five batches of Liushenqu tablets (210901, 211203, 220603, 220702, and 220701) were tested according to the method for determining the characteristic chromatograms of the standard decoction of Liushenqu. The results are shown in Tables 26-27 below. The results show that the relative retention time (RSD) of each characteristic peak in each batch is less than 2%, and all are within the range of ±10% of the mean.

[0222] Table 26 Results of relative retention time determination of Liushenqu (a traditional Chinese medicine) slices' characteristic chromatograms.

[0223]

[0224] Table 27. Results of relative peak area determination of characteristic chromatogram of (stir-fried) Liu Shen Qu standard decoction.

[0225]

[0226] Example 8

[0227] This embodiment uses the characteristic spectrum determination of the standard decoction of Liu Shen Qu (a traditional Chinese medicine).

[0228] The characteristic chromatograms of multiple batches of Liushenqu standard decoction were determined according to the established method for [characteristic chromatogram] (210901Y, 211203Y, 220603Y, 220702Y, 220701Y). The results are shown in Tables 28-29 below. The results show that the relative retention time (RSD) of each characteristic peak in each batch is less than 2%, and all are within ±10% of the mean.

[0229] Table 28 Results of relative retention time determination for the characteristic chromatograms of Liushenqu standard decoction

[0230]

[0231] Table 29. Results of relative peak area determination of characteristic chromatogram of (stir-fried) Liu Shen Qu standard decoction.

[0232]

[0233] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic spectrum of Liushenqu and its pharmaceutical preparations, characterized in that, This includes the step of performing ultra-high performance liquid chromatography detection on the test solution of Liushenqu and / or Liushenqu drug preparation; Chromatographic conditions included: a Waters ACQUITYUPLC HSS T3 column, 2.1 × 100 mm, 1.8 μm; and gradient elution using methanol as mobile phase A and 0.1% formic acid as mobile phase B, according to the following program: 0-8 min, A:B ratio was 0 → 3%; 100% → 97%; 8-15 min, A:B changed from 3% to 13%; 97% changed from 97% to 87%; 15-30 min, A:B ratio changed from 13% to 38%; 87% to 62%. The preparation method of the test sample solution includes: precisely adding the test sample to the first solvent and mixing, ultrasonically treating the mixture, taking the supernatant and treating it with a macroporous adsorption resin, eluting it with a second solvent, evaporating it to dryness and redissolving it to obtain the final product; The first solvent is water; The second solvent is a 40%-50% methanol solution; The macroporous adsorption resin includes macroporous adsorption resin D101; The reference standards used in the method for constructing the characteristic spectrum of the Liushenqu and its pharmaceutical preparations include: uridine, adenosine, guanosine, 5-hydroxymethylfurfural, L-tryptophan, vanillic acid, ferulic acid, and shampooside. The chromatographic conditions include: column temperature of 30°C and detection wavelength of 270 nm.

2. The method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations according to claim 1, characterized in that, The ultra-high performance liquid chromatography detection step also includes the following chromatographic conditions: a flow rate of 0.3 ml / min.

3. The method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations according to claim 1, characterized in that, The method also includes the step of preparing a reference solution of the control medicinal material, specifically including: taking the Liu Shen Qu control medicinal material and accurately adding it to the first solvent and mixing it, collecting the extract after heating and reflux, filtering it, taking the supernatant for macroporous adsorption resin treatment, eluting it with the second solvent, evaporating it to dryness and redissolving it to obtain the solution.

4. The method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations according to claim 1, characterized in that, The method further includes the steps of preparing a reference solution and constructing a characteristic chromatogram of the reference solution based on the ultra-high performance liquid chromatography. The preparation method of the reference solution includes: taking the reference standard and adding it to a second solvent and mixing them to obtain the solution.

5. The method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations according to claim 1, characterized in that, The Liushenqu pharmaceutical preparations include Liushenqu formula granules, Liushenqu decoction pieces, or Liushenqu standard decoction.

6. The method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations according to claim 1, characterized in that, The characteristic spectrum has nine characteristic peaks, with peak 6 designated as the S peak. The relative retention times of the other characteristic peaks relative to peak 6 are within ±10% of a specified value. The specified values ​​for the nine characteristic peaks are: peak 1: 0.25, peak 2: 0.47, peak 3: 0.55, peak 4: 0.58, peak 5: 0.83, peak 7: 1.31, peak 8 ...47, peak 2: 0.47, peak 3: 0.47, peak 4: 0.58, peak 5: 0.83, peak 7: 1.31, peak 8: 0.47, peak 2: 0.47 1.75 and peak 9: 1.

88.

7. The application of the method for constructing the characteristic spectrum of Liushenqu and its pharmaceutical preparations as described in any one of claims 1-6 in the field of quality testing of Liushenqu and its pharmaceutical preparations.

8. A quality testing method for Liushenqu and its pharmaceutical preparations, characterized in that, The method includes the steps of constructing the feature map and the reference feature map according to any one of claims 1-6, and the step of comparing the feature map with the reference feature map.

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