Integrated detection method for quantification and identification of free anthraquinones in rhubarb

Abstract

The present application belongs to the technical field of medicinal material detection and analysis, and particularly relates to an integrated detection method for free anthraquinone quantification and authenticity identification of rhubarb medicinal materials. The present application solves the problem of simultaneous free anthraquinone quantification and authenticity identification of rhubarb by determining rhubarb mixed control solution and test solution respectively through one-dimensional and two-dimensional reverse phase chromatography. Compared with the traditional HPLC and TLC combined method, the present application simplifies the pretreatment, improves the sensitivity and specificity, shortens the analysis time, avoids the use of volatile toxic reagents, and the results are more intuitive. The methodological verification shows that the present application has good specificity, precision, accuracy and linear relationship, and is suitable for quality control and authenticity identification of rhubarb.

Description

Technical Field

[0001] This invention belongs to the field of medicinal material detection and analysis technology, specifically involving an integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb. Background Technology

[0002] Rhubarb is a representative medicinal species of the Polygonaceae family, including Rheum palmatum (Rheum palmatum). Rheum palmatum L.), Tangut rhubarb ( Rheum tanguticum Maxim. ex Balf.) and medicinal rhubarb ( Rheum officinale Rhubarb (including Baill.) is one of the four major traditional Chinese medicines. With the continuous growth in market demand for rhubarb, adulteration has become increasingly common. Due to the multi-source characteristics of rhubarb, genuine rhubarb and its adulterants (such as North China rhubarb and Hetao rhubarb) are extremely similar in appearance and microscopic features, making them difficult to distinguish and seriously affecting the safety and efficacy of clinical use. Therefore, accurate identification of genuine and adulterated rhubarb is crucial to ensuring the safety of clinical medication.

[0003] Emodin is a stilbene derivative, mainly found in counterfeit rhubarb, and is a key indicator for identifying genuine rhubarb. In the 2020 edition of the Chinese Pharmacopoeia, emodin is listed as an inspection item, detected using thin-layer chromatography (TLC) for qualitative identification of rhubarb. Besides authenticity identification, the content of active ingredients in rhubarb is also an important indicator of its quality. The 2020 edition of the Chinese Pharmacopoeia specifies methods for determining the content of bound and free anthraquinones in rhubarb. The pretreatment methods differ, with bound anthraquinones requiring more complex pretreatment, but both are determined using liquid chromatography (LC) under the same chromatographic conditions.

[0004] The above analytical method has the following shortcomings:

[0005] (1) Sample processing is complicated and inefficient: The determination of free anthraquinone content in rhubarb and the identification of authenticity in the Chinese Pharmacopoeia require different sample extraction operations, which increases the workload and reagent costs.

[0006] (2) Poor environmental performance: The developing solvent used in thin-layer chromatography contains highly volatile and toxic reagents such as acetone and ethyl formate, which are very harmful to the environment.

[0007] (3) Low integration of analytical methods: The Chinese Pharmacopoeia uses liquid chromatography and thin-layer chromatography to determine the content and identify the authenticity, which requires operation on different instruments and the process is complicated.

[0008] (4) If the five free anthraquinones in the Chinese Pharmacopoeia are used to detect rhein, due to its high polarity, the peak will be too early under these conditions, and it cannot be effectively retained on the chromatographic column. The separation effect with adjacent substances is poor, and it cannot be determined at the same time.

[0009] (5) The sensitivity and specificity of qualitative methods are limited: The thin-layer chromatography method used in the Chinese Pharmacopoeia has low sensitivity for detecting rhein, making it difficult to detect trace adulterants. Furthermore, the specificity of thin-layer chromatography is easily affected by various factors, which limits its application in identifying genuine and counterfeit medicinal materials.

[0010] (6) Complex data parsing: Existing detection methods will generate multiple data combinations, which increases the difficulty of data parsing. Summary of the Invention

[0011] This invention aims to address the shortcomings of existing methods for quality control of rhubarb medicinal materials. It provides an integrated and efficient detection scheme for the quantification of free anthraquinones in rhubarb and the identification of authenticity based on two-dimensional liquid chromatography. This scheme enables the determination of the content of five free anthraquinones in rhubarb and the identification of authenticity of the medicinal material in a single pretreatment and single injection, thereby improving the sensitivity and specificity of detection, shortening the analysis time, reducing the complexity of data processing, and reducing reagent toxicity.

[0012] The integrated detection method for quantitative determination and authenticity identification of free anthraquinones in rhubarb described in this invention uses a two-dimensional liquid chromatography method, which includes one-dimensional liquid chromatography analysis and two-dimensional liquid chromatography analysis.

[0013] The one-dimensional liquid chromatography (HPLC) analysis conditions were as follows: ShimNex CS C18, 4.6 × 250 mm, 5 μm; mobile phase: 0.1% phosphoric acid solution-methanol, isocratic elution; flow rate: 1.0 ± 0.05 mL / min; column temperature: 25-40℃; injection volume: 5-20 μL; detection wavelength: 254 ± 5 ​​nm; and the volume ratio of methanol in the mobile phase was 83%-90% in the one-dimensional HPLC analysis conditions.

[0014] The two-dimensional liquid chromatography analysis conditions were as follows: Shim-pack GIS C18, 4.6×250mm, 5μm; mobile phase A was water, mobile phase B was acetonitrile, gradient elution; flow rate was 1.0±0.05mL / min; column temperature was 25-40℃; detection wavelength was 320±5nm.

[0015] The gradient elution program for the two-dimensional liquid chromatography analysis is as follows: 0~15 min, 20% B, 15~30 min, 20%~35% B, 30~34 min, 35%~95% B, 34~39 min, 95% B, 39~39.01 min, 95%~20% B, 39.01~40 min, 20% B.

[0016] The specific steps are as follows:

[0017] (1) The mixed reference solution, the test solution and the mixed test solution were respectively introduced into the full-spectrum two-dimensional liquid chromatography system. After the injection, the highly polar substances were introduced into the sample loop for collection, and the two-dimensional C18 column was equilibrated at the same time.

[0018] (2) After the high polarity substances have been collected, the six-way valve switches the valve position from state 1 to state 0. The low polarity substances are retained and separated on the one-dimensional chromatographic column, while the high polarity substances are focused and enriched on the column head of the two-dimensional chromatographic column.

[0019] (3) After the one-dimensional liquid phase analysis is completed, the position of the six-way valve is switched again from state 0 to state 1. At the same time, the detection wavelength is switched from 254±5nm to 320±5nm. Highly polar substances are retained and separated on the two-dimensional liquid chromatography.

[0020] State 0 is a one-dimensional chromatographic column connected to a detector, and state 1 is a one-dimensional chromatographic column connected to a quantitative loop.

[0021] The preparation steps of the test sample solution of the present invention are as follows: take the test sample powder, accurately weigh it, add methanol, weigh it, heat it under reflux, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0022] Specifically: Take 0.5g of the test sample powder, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, heat under reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the test sample.

[0023] The preparation steps of the mixed reference solution of the present invention are as follows: accurately weigh each reference standard and add methanol to prepare reference solution respectively; accurately measure each reference solution and dilute it with methanol to obtain the mixed reference solution.

[0024] Specifically: Accurately weigh the reference standards for aloe-emodin, rhein, emodin, chrysophanol, emodin methyl ether, and emodin-modin, and dissolve them in methanol to prepare reference solutions containing 80 μg each of aloe-emodin, rhein, emodin, and chrysophanol, 40 μg of emodin methyl ether, and 400 μg of emodin-modin per ml. Accurately measure the above reference solutions and dilute them with methanol to prepare a mixed reference solution containing 16 μg each of aloe-emodin, rhein, emodin, and chrysophanol, 8 μg of emodin methyl ether, and 10 μg of emodin-modin per ml.

[0025] The preparation steps of the mixed test solution of the present invention are as follows: take appropriate amounts of each positive and negative test sample, mix them evenly in proportion, weigh them accurately, add methanol, weigh them, heat under reflux, cool them, weigh them again, make up the weight loss with methanol, shake well, filter them, and take the filtrate to obtain the solution.

[0026] Specifically: Take appropriate amounts of commercially available rhubarb powder and North China rhubarb reference powder in a ratio of 97:3, mix them evenly, take about 0.5g of the powder, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, heat under reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the final product.

[0027] The integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb described in this invention can be applied to the quality control of rhubarb.

[0028] The present invention has the following beneficial effects:

[0029] (1) Simplified sample processing procedure: The present invention can be used for the quantitative analysis of five free anthraquinones in rhubarb and the qualitative detection of rhein in one sample preparation. There is no need to extract and prepare samples separately for authenticity identification. This effectively simplifies the operation process, reduces the workload of pretreatment, reduces sample loss, and greatly improves the analysis efficiency.

[0030] (2) Enhanced sensitivity and specificity: The present invention uses liquid chromatography to identify the authenticity of rhubarb, which can better separate and detect the components in the sample, and can also accurately identify trace amounts of rhein (such as 0.2% adulteration), significantly improving the sensitivity and specificity of detection, and can more accurately identify the authenticity of rhubarb medicinal materials, reducing the risk of misjudgment.

[0031] (3) Improved environmental friendliness: The present invention uses liquid chromatography, which avoids the use of highly volatile toxic reagents, reduces experimental risks, and protects the health of operators.

[0032] (4) Shortened analysis time: This invention combines the separation and quantification of five free anthraquinones by one-dimensional C18 reversed-phase chromatography and the qualitative analysis of emodin by two-dimensional C18 chromatography through the integrated design of two-dimensional liquid chromatography, and realizes integrated detection. It enables the simultaneous quantitative and qualitative analysis. The quantitative analysis of five free anthraquinones in one-dimensional chromatography and the qualitative (or even quantitative) analysis of emodin in two-dimensional chromatography can be completed within 40 minutes on one chromatographic system. Compared with traditional methods, the overall analysis time is greatly shortened, and results can be quickly provided for the quality control of rhubarb medicinal materials.

[0033] (5) Simplified data processing: This invention uses a two-dimensional liquid chromatography system and wavelength switching function to present the quantitative analysis of five free anthraquinones and the qualitative analysis of rhein on a single chromatogram, avoiding the complex processing of multiple data, simplifying the data analysis process, improving data processing efficiency, and facilitating a more intuitive evaluation of the quality of rhubarb medicinal materials. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of a full-spectrum two-dimensional liquid chromatography system (top: state 1, bottom: state 0), where: 1 is a one-dimensional liquid chromatography pump A; 2 is a one-dimensional liquid chromatography pump B; 3 is a two-dimensional liquid chromatography pump A; 4 is a two-dimensional liquid chromatography pump B; 5 is an autosampler; 6 is a one-dimensional chromatographic column; 7 is a two-position six-way valve (left); 8 is a two-position six-way valve (right); 9 is a two-dimensional chromatographic column; 10 is a sample loop; and 11 is a detector. State 1 indicates a one-dimensional chromatographic column connected to a quantitative loop, and state 0 indicates a one-dimensional chromatographic column connected to a detector.

[0035] Figure 2 The superimposed chromatograms of the reference standard and the test sample solution under two-dimensional liquid chromatography (6 components of mixed reference standard, commercially available rhubarb slices, and commercially available rhubarb adulterated with 3% North China rhubarb reference material).

[0036] Figure 3 The chromatogram of commercially available rhubarb adulterated with 0.2% North China rhubarb reference material;

[0037] Figure 4 Calibration curve for emodin;

[0038] Figure 5 A peak purity diagram of phytoestrogen in the mixed test solution;

[0039] Figure 6 MS1 scan mass spectrum;

[0040] Figure 7 The result is the molecular formula prediction.

[0041] Figure 8 This is the result of a structured database search. Detailed Implementation

[0042] This experiment used the Shimadzu full-spectrum two-dimensional liquid chromatography system, specifically configured as follows:

[0043] .

[0044] Example 1

[0045] An integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb, the specific steps of which are as follows:

[0046] 1) Preparation of mixed reference solution: Accurately weigh appropriate amounts of aloe-emodin reference standard, rhein reference standard, emodin reference standard, chrysophanol reference standard, emodin methyl ether reference standard, and physcion reference standard, and add methanol to prepare reference solutions containing 80 μg of aloe-emodin, rhein, emodin and chrysophanol, 40 μg of emodin methyl ether, and 400 μg of physcion per ml; accurately measure appropriate amounts of the above reference solutions and dilute with methanol to prepare mixed reference solutions containing 16 μg each of aloe-emodin, rhein, emodin, and chrysophanol, 8 μg of emodin methyl ether, and 10 μg of physcion per ml.

[0047] 2) Preparation of the test solution: Take about 0.5g each of genuine rhubarb (medicinal rhubarb) reference powder, counterfeit North China rhubarb reference powder, and commercially available rhubarb slices powder, accurately weigh them, place them in stoppered conical flasks, accurately add 25ml of methanol, weigh them, heat under reflux for 1 hour, cool them, weigh them again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0048] 3) Preparation of mixed test solution: Take appropriate amounts of commercially available rhubarb powder and North China rhubarb reference powder in a ratio of 97:3, mix them evenly, take about 0.5g of the powder, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, weigh it, heat under reflux for 1 hour, cool it, weigh it again, make up the weight loss with methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0049] 4) Determination method: Accurately pipette 10 μl each of the mixed reference solution, the test solution, and the mixed test solution, and inject them into the liquid chromatograph for determination. The liquid chromatographic conditions are shown in Table 1-3, and the full-spectrum two-dimensional chromatographic system is as follows: Figure 1 As shown.

[0050] Table 1. Time program for two-dimensional liquid chromatography (initial mobile phase B is 20%)

[0051] .

[0052] Table 2 Time program for two-dimensional liquid chromatography (initial mobile phase B is 20%)

[0053] .

[0054] Table 3 Other Time Procedures

[0055] .

[0056] Note: The column oven valve command "0" is for one-dimensional chromatography column with detector, and "1" is for one-dimensional chromatography column with quantitative loop.

[0057] Specifically, pipette 10 μL each of the mixed reference solution, the test solution, and the mixed test solution into the full-spectrum two-dimensional liquid chromatography system. After injection, if... Figure 1 As shown in the schematic diagram above, highly polar substances, including emodin, are not retained by the one-dimensional chromatographic column and are introduced into the sample loop for collection along with the mobile phase, while the two-dimensional C18 column is equilibrated.

[0058] Once the highly polar substances have been collected, such as Figure 1 As shown in the schematic diagram below, the six-way valve switches the valve position from state 1 to state 0. After that, low polar substances, including five free anthraquinones, are retained and separated on the one-dimensional chromatographic column, realizing the quantitative analysis of the five free anthraquinones. At the same time, high polar substances are enriched by column-head focusing on the two-dimensional chromatographic column.

[0059] The one-dimensional liquid phase analysis was completed after 20 minutes, as follows: Figure 1 As shown in the diagram above, the position of the six-way valve is switched again from state 0 to state 1, and the detection wavelength is switched from 254nm to 320nm. At this time, highly polar substances such as emodin are retained and separated on the two-dimensional liquid chromatography, realizing the qualitative detection and even quantitative analysis of emodin.

[0060] The overlay graph of the chromatograms of the mixed reference solution, the test solution, and the mixed test solution under these conditions is shown below. Figure 2 As shown in the figure, the free anthraquinone compounds aloe-emodin, rhein, emodin, chrysophanol, and emodin methyl ether in the three solutions eluted on one-dimensional chromatography, and each peak was well separated from adjacent peaks, allowing for quantitative analysis of the five free anthraquinone compounds. The qualitative identification marker emodin eluted on two-dimensional chromatography and was well separated from adjacent peaks. Emodin was not detected in the raw rhubarb decoction sample solution, but the emodin peak was more obvious in the raw rhubarb decoction sample adulterated with 3% North China rhubarb, indicating that a small amount of North China rhubarb adulteration added to genuine rhubarb can be accurately detected.

[0061] Example 2 Methodological Investigation

[0062] 1. Exclusivity

[0063] When methanol was used as a blank solvent, no obvious chromatographic peaks were observed at the elution positions of the five free anthraquinones and emodin in the blank solvent, which did not interfere with the qualitative and quantitative detection.

[0064] 2. Precision

[0065] The mixed reference solution was measured six times, and the results are shown in Table 4. The retention time RSD of the six test compounds was between 0.051% and 0.093%, and the peak area RSD was between 0.040% and 0.504%, indicating that the method has good precision.

[0066] Table 4 Precision Test Results

[0067] .

[0068] 3. Calibration curve

[0069] Take appropriate amounts of commercially available rhubarb powder and North China rhubarb reference powder, and mix them thoroughly in ratios of 99.8:0.2, 99.5:0.5, 99:1, 97:3, and 95:5, respectively. Accurately weigh approximately 0.5g of each powder mixture and place them in separate stoppered conical flasks. Accurately add 25ml of methanol, weigh the flasks, heat under reflux for 1 hour, cool, and weigh again. Make up the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the calibration curve solution. Inject 10μL of each solution for analysis. The chromatogram of the adulterated 0.2% North China rhubarb reference material is shown below. Figure 3 As shown, the chromatographic peak of emodin is relatively obvious, indicating that this method has high sensitivity for adulteration identification. A calibration curve was constructed with the adulteration ratio of the adulterant, North China rhubarb, as the x-axis and the peak area of ​​emodin as the y-axis, as shown in the figure. Figure 4 As shown in the figure. The results showed that the peak area of ​​emodin was linearly related to the proportion of adulterated rhubarb, with a regression equation of Y=535398X-2764.96 and a correlation coefficient R of 0.9996. The backread values ​​of each linear point were between 93.2% and 109.6% of the theoretical values.

[0070] 4. Accuracy

[0071] The determination of free anthraquinones in rhubarb according to the Chinese Pharmacopoeia uses the traditional one-dimensional liquid chromatography method. This experiment innovatively uses two-dimensional liquid chromatography to quantitatively analyze five free anthraquinones in rhubarb. To evaluate the accuracy of the quantitative analysis using the full-spectrum two-dimensional liquid chromatography method, we took medicinal rhubarb reference material, commercially available rhubarb slices, North China rhubarb reference material, and a mixed sample (3%) of commercially available rhubarb and North China rhubarb reference material as examples. The full-spectrum two-dimensional liquid chromatography method was compared with the statutory test method in the Chinese Pharmacopoeia. The results are shown in Table 5. The relative deviation of the total amount of the five free anthraquinones in the four samples was between -2.17% and -0.40%, indicating that there was no significant difference between the two methods.

[0072] Table 5. Comparison of quantitative results of total free anthraquinones using two different analytical methods

[0073] .

[0074] 5. Durability Assessment

[0075] This experiment investigated the effects of one-dimensional mobile phase ratio changes, injection volume, flow rate, column temperature, and detection wavelength on separation and detection. The specific factors investigated are shown in Table 6.

[0076] Table 6 Results of different factors examined

[0077] .

[0078] Experimental data showed that, under the standard chromatographic system, the following conditions had no significant impact on the separation and detection of the analyte compounds: a methanol content of 83%–90% in the one-dimensional mobile phase (corresponding to a methanol-0.1% phosphoric acid solution ratio of 83:17 to 90:10); an injection volume of 5–20 μL; a flow rate variation of ±0.05 mL / min in both one-dimensional and two-dimensional chromatography; a column temperature variation of -5℃ to +10℃; and a detection wavelength variation of ±5 nm in both one-dimensional and two-dimensional chromatography. However, when the one-dimensional mobile phase ratio was 80:20, emodin methyl ether failed to elute successfully before the end of the one-dimensional analysis time. Therefore, in this system, the methanol-0.1% phosphoric acid solution ratio (80:20) should not be used as the one-dimensional mobile phase.

[0079] 6. Peak purity test

[0080] Taking the mixed reference standard and mixed test sample solutions as examples, a PDA diode array detector was used for detection in the wavelength range of 190-800 nm to investigate the peak purity of five free anthraquinone compounds and emodin under two-dimensional liquid chromatography. The peak purity detection results are shown in Table 7, and the peak purity test chromatogram of the mixed test sample solution is shown in... Figure 5 As shown, the minimum peak purity index of the five free anthraquinone quantitative compounds and the identification marker emodin in the mixed reference standard and mixed test solution were all greater than 0. That is, from the perspective of spectral similarity analysis, the chromatographic peaks of the six compounds were all single chromatographic peaks and did not contain other substances. This method can provide a reliable basis for the quantitative and authenticity identification analysis of the five free anthraquinone compounds in rhubarb.

[0081] Table 7 Peak purity test results

[0082] .

[0083] 7. Qualitative analysis of rhein from phytoestrogen

[0084] To further confirm that the liquid collected within the quantitative loop can be separated by two-dimensional liquid chromatography (2D-LC) for qualitative analysis of emodin, in addition to comparing retention time and spectral similarity with the reference standard, this experiment also combined full-spectrum 2D-LC with quadrupole time-of-flight high-resolution mass spectrometry (LCMS-9050) to identify the "emodin" separated on the 2D chromatogram (retention time Rt approximately 29 min). The LCMS-9050 was used to acquire MS1 data of the components collected in 2D-LC in both positive and negative modes. The results showed that a strong signal of m / z 421.14898 was detected in the positive ion mode (e.g., ...). Figure 6 As shown in the figure, the molecular weight of the compound to be tested is estimated to be 420. Based on the accurate molecular weight and isotopic abundance ratio, the possible molecular formula is predicted using the Predict function in LabSolutions Insight Explore. The prediction results are shown in the figure. Figure 7 As shown. The results indicate that the possible molecular formula of the analyte is C. 21 H 24 O9, [M+H] + The mass-to-charge ratio error was -0.784 ppm, and the isotope distribution matching score was 98.84.

[0085] The molecular formula was sent to the software's built-in Chemspider or PubChem public database for structural search. Simultaneously, the Assign function in Labsolutions Insight Explore software was used for secondary mass spectrometry assignment. The search and assignment results are as follows: Figure 8 As shown, the substance with the best matching in the secondary mass spectrum is rhaponticin, with an Assign matching score of 90.1. This further confirms, using high-resolution mass spectrometry, that the compound with a retention time of approximately 29 min is rhaponticin.

Claims

1. An integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb, characterized in that, Two-dimensional liquid chromatography was used to determine the mixed reference solution and the test solution, respectively. The two-dimensional liquid chromatography method includes one-dimensional liquid chromatography analysis and two-dimensional liquid chromatography analysis. The one-dimensional liquid chromatography analysis conditions were as follows: ShimNex CS C18, 4.6×250mm, 5μm; mobile phase: 0.1% phosphoric acid solution-methanol, isocratic elution; flow rate: 1.0±0.05mL / min; column temperature: 25-40℃; injection volume: 5-20μL; detection wavelength: 254±5nm. The two-dimensional liquid chromatography analysis conditions were as follows: Shim-pack GIS C18, 4.6×250mm, 5μm; mobile phase A was water, mobile phase B was acetonitrile, gradient elution; flow rate was 1.0±0.05mL / min; column temperature was 25-40℃; detection wavelength was 320±5nm. The specific steps are as follows: (1) The mixed reference solution, the test solution and the mixed test solution were respectively introduced into the full-spectrum two-dimensional liquid chromatography system. After the injection, the highly polar substances were introduced into the sample loop for collection, and the two-dimensional C18 column was equilibrated at the same time. (2) After the high polarity substances have been collected, the six-way valve switches the valve position from 1 to 0. The low polarity substances are retained and separated on the one-dimensional chromatographic column, while the high polarity substances are focused and enriched on the column head of the two-dimensional chromatographic column. (3) After the one-dimensional liquid phase analysis is completed, the position of the six-way valve is switched again from 0 to 1, and the detection wavelength is switched from 254±5nm to 320±5nm. Highly polar substances are retained and separated on the two-dimensional liquid chromatography. The "0" represents a one-dimensional chromatographic column connected to a detector, and the "1" represents a one-dimensional chromatographic column connected to a quantitative loop.

2. The integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb according to claim 1, characterized in that, In one-dimensional liquid chromatography analysis, the volume ratio of methanol in the mobile phase is 83%-90%.

3. The integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb according to claim 1, characterized in that, The gradient elution program for the two-dimensional liquid chromatography analysis is as follows: 0~15 min, 20% B, 15~30 min, 20%~35% B, 30~34 min, 35%~95% B, 34~39 min, 95% B, 39~39.01 min, 95%~20% B, 39.01~40 min, 20% B.

4. The integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb according to claim 1, characterized in that, The preparation steps of the test sample solution are as follows: take the test sample powder, weigh it accurately, add methanol, weigh it, heat it under reflux, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

5. The integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb according to claim 1, characterized in that, The preparation steps of the mixed reference solution are as follows: accurately weigh aloe-emodin reference standard, rhein reference standard, emodin reference standard, chrysophanol reference standard, emodin methyl ether reference standard, and physcion reference standard, and add methanol to prepare reference solutions respectively; accurately measure an appropriate amount of each reference solution and dilute it with methanol to obtain the mixed reference solution.

6. The integrated detection method for quantitative analysis and authenticity identification of free anthraquinones in rhubarb as described in claim 1 is applied to the quality control of rhubarb.

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