A quality testing method for a purifying and acid-lowering compound

Abstract

The present application belongs to the field of medicine, and particularly relates to a quality detection method of a Shengqing Jiangsuan mixture. The present application firstly establishes a characteristic spectrum method for simultaneously detecting 10 main active components (paeoniflorin, puerarin, gentiopicroside, loganin acid, astilbin, benzoylpaeoniflorin, resveratrol, rhein, pachymaran A and pachymaran B) in the mixture, overcomes the limitation of single component control quality, and can more comprehensively and objectively evaluate the overall quality of the medicine. Through optimizing the sample pretreatment process and the chromatographic gradient elution conditions, the present application effectively eliminates the interference of the complex matrix, and ensures the good separation of each component. The method has strong specificity, no interference of negative, and can accurately identify the target component.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and specifically relates to a quality testing method for a compound preparation for raising urination and lowering acidity. Background Technology

[0002] Hyperuricemia (HUA) and the resulting gout have become common metabolic diseases worldwide. Its incidence is rising annually and showing a trend towards affecting younger people, severely impacting patients' quality of life. Modern medicine believes that the essence of this disease is purine metabolism disorder and / or reduced uric acid excretion, leading to elevated blood uric acid levels. Uric acid crystals then deposit in joints, soft tissues, kidneys, and other areas, causing pathological changes such as acute arthritis, tophi, and uric acid kidney stones.

[0003] Currently, first-line drugs for the clinical treatment of hyperuricemia and gout in Western medicine mainly include allopurinol and febuxostat, which inhibit uric acid production, and benzbromarone, which promotes uric acid excretion. While these drugs have some efficacy in controlling acute symptoms and lowering blood uric acid levels, their limitations are also quite prominent: First, chemical drugs are often accompanied by varying degrees of side effects, such as liver and kidney damage, gastrointestinal reactions, and skin allergies, leading to poor patient tolerance and questionable safety with long-term use; second, existing treatments mostly focus on the single target of "lowering uric acid," failing to effectively improve systemic symptoms commonly experienced by patients, such as dizziness, fatigue, heaviness in the limbs, and loss of appetite, thus failing to meet the overall conditioning needs of patients; third, blood uric acid levels tend to rebound after discontinuation of medication, making it difficult to fundamentally stabilize and regulate metabolic status.

[0004] From the perspective of traditional Chinese medicine, hyperuricemia and gout fall under the categories of "Bi syndrome," "turbidity and stasis Bi syndrome," and "arthritis." Traditional Chinese medicine theory holds that the root cause lies in spleen deficiency and impaired transport function. The spleen governs transportation and transformation, is the source of qi and blood, and is also the pivot of qi's ascending and descending. Spleen deficiency weakens the body's ability to transport and transform fluids, leading to the internal generation of dampness; clear yang fails to ascend, and turbid yin fails to descend, causing dampness and turbidity to obstruct the meridians, stagnate qi and blood, and over time transform into blood stasis and toxins, flowing into the joints, causing redness, swelling, heat, and pain. Therefore, its core pathogenesis can be summarized as spleen deficiency as the root cause, and dampness and turbidity obstructing the meridians and stagnating blood stasis and toxins as the manifestations. Clinically, it commonly presents with a series of symptoms such as dizziness and fatigue (due to failure of clear yang to ascend), heaviness in the limbs (due to dampness and turbidity), a swollen tongue with a white and greasy coating (a sign of internal dampness and turbidity), and joint pain (due to obstructed meridians).

[0005] The Shengqing Jiangsuan Mixture consists of eleven ingredients: Pueraria lobata, Cinnamomum cassia, Paeonia lactiflora, Chicory, Coix lacryma-jobi, Smilax glabra, Dioscorea hypoglauca, Gentiana macrophylla, Polygonum cuspidatum, Lysimachia christinae, and Lilium brownii. The prescription applies the theory of "stagnation damaging the spleen and stomach," treating the condition from the perspective of the spleen. Based on traditional gout treatments, it adds Cinnamomum cassia, Paeonia lactiflora, Pueraria lobata, Coix lacryma-jobi, and Lilium brownii. Cinnamomum cassia is yang, and Paeonia lactiflora is yin, forming a foundational formula for strengthening the spleen and stomach. The yin and yang of these two ingredients harmonize, restoring the spleen and stomach's digestive function. Furthermore, "for those suffering from phlegm and fluid retention, warm medicines should be used to harmonize them." Water and phlegm are transformed when warmed. Cinnamomum cassia warms the spleen yang, transforms spleen dampness, and also warms the meridians and unblocks the channels, transforming dampness in the meridians. Paeonia lactiflora removes water retention and relieves pain. Together, they strengthen the spleen, promoting the upward movement of clear qi and the downward movement of turbid qi, forming the basis for treating internal dampness and turbidity. The combination of kudzu root and cinnamon twig enhances the spleen-strengthening and qi-regulating effects of this formula. Kudzu root is known for its ability to treat various types of numbness and detoxify, including acid toxicity and dampness obstructing the limbs and joints. The combination of kudzu root and cinnamon twig further enhances the spleen-strengthening and qi-regulating effects. Coix seed is effective in removing dampness and strengthening the spleen, and is highly effective in treating beriberi. Lily bulb promotes the removal of dampness and facilitates urination and defecation. This combination of herbs utilizes traditional gout-relieving properties to improve intestinal function and enhance the intestines' ability to eliminate turbidity. It alleviates symptoms such as sticky stools and constipation in patients with hyperuricemia and gout, accelerates uric acid metabolism, reduces secondary uric acid absorption in the intestines, lowers blood uric acid levels, prevents and treats gout, and reduces the burden on the kidneys to excrete uric acid. The challenge in detecting this formula lies in the difficulty of optimizing chromatographic conditions for simultaneous detection of multiple components. It requires the simultaneous detection of more than 10 components under single chromatographic conditions, with significant differences in polarity, solubility, and response values. Gradient elution procedures are also very complex, with frequent changes in the mobile phase ratio, and high requirements for column efficiency and system stability. It is necessary to ensure that all target peaks are completely separated within a reasonable time, and that the theoretical plate number, resolution, and peak shape all meet the requirements. Summary of the Invention

[0006] The purpose of this invention is to provide a quality testing method for Shengqing Jiangsuan compound, thereby improving the efficacy and safety of Shengqing Jiangsuan compound in clinical applications by establishing quality control standards.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] A quality testing method for a purgative and acid-lowering compound includes the following steps:

[0009] Step 1: Preparation of reference solution

[0010] Weigh out the reference standards paeoniflorin, puerarin, gentiopicrin, loganic acid, astilbene, benzoylpaeoniflorin, resveratrol, rhein, squalene A, and squalene B, and add methanol to prepare a mixed solution containing 60 µg paeoniflorin, 45 µg puerarin, 45 µg gentiopicrin, 30 µg loganic acid, 40 µg astilbene, 20 µg benzoylpaeoniflorin, 12 µg resveratrol, 25 µg rhein, 30 µg squalene A, and 30 µg squalene B per mL. This is the reference solution.

[0011] Step 2: Preparation of the test solution

[0012] Accurately measure 10 mL of the serotonin-lowering and acid-lowering compound, add 1 mol / L phosphate buffer to adjust the pH to 5.0, then add the compound enzymatic hydrolysate and incubate at 45°C for 45 minutes. After cooling, adjust the pH of the hydrolysate to 2.5 with 10% hydrochloric acid solution, then transfer it to a separatory funnel. Degrease the hydrolysate adjusted to pH 2.5 with 10% hydrochloric acid solution using cyclohexane and discard the organic phase. Extract the aqueous phase three times with ethyl acetate by shaking. Combine the ethyl acetate extracts and evaporate under reduced pressure at 45°C to dryness to obtain the residue. Dissolve the residue thoroughly in 10 mL of methanol to obtain a preliminary purified solution. Load the preliminary purified solution at a flow rate of 1 mL / min onto an Oasis HLB solid-phase extraction column pre-activated with 5 mL of methanol and 5 mL of water. After loading, rinse with 5 mL of 5% methanol aqueous solution and discard. Then rinse with 5 mL of... A 40% methanol aqueous solution was used for initial elution and discarded. Finally, 8 mL of 85% methanol aqueous solution containing 0.1% formic acid (by volume) was used to elute the target component, and all eluent was collected. The collected eluent was concentrated to near dryness by nitrogen blowing at 50°C to obtain a residue. The residue was then reconstituted by ultrasonication with 1.0 mL of methanol-acetonitrile mixed solvent. The reconstituted solution was filtered through a 0.22 μm nylon microporous membrane, and the resulting filtrate was the test solution.

[0013] Step 3: Determination Method

[0014] Inject 10 μL of the reference solution and the test solution into the liquid chromatograph and determine their contents.

[0015] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile:methanol (v / v) at a ratio of 78:22 was used as mobile phase A, and 0.1% phosphoric acid solution containing 0.1g sodium dodecyl sulfonate per 100mL was used as mobile phase B for gradient elution; the column temperature was 30℃; the UV detector wavelength was 230nm; the theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 5000; the gradient elution conditions were as follows:

[0016] From 0 to 10 min, the volume ratio of mobile phase A to phase B changed from 4:96 to 12:88.

[0017] 10~18min, the volume ratio of mobile phase A: phase B changes from 12:88 to 14:86;

[0018] 18~30min, the volume ratio of mobile phase A: phase B changes from 14:86 to 20:80;

[0019] 30~45min, the volume ratio of mobile phase A: phase B changes from 20:80 to 25:75;

[0020] 45~75 min, the volume ratio of mobile phase A: phase B changes from 25:75 to 60:40;

[0021] 75~76 min, the volume ratio of mobile phase A: phase B changes from 60:40 to 4:96;

[0022] 76~85min, mobile phase A: phase B, volume ratio 4:96;

[0023] Step 4: Generate a reference characteristic chromatogram: Select the chromatographic peaks that are present in the chromatograms of different batches of the Shengqing Jiangsuan compound as common peaks, and use the average value calculation method to generate a reference characteristic chromatogram of the Shengqing Jiangsuan compound.

[0024] Furthermore, in step two, the compound enzymatic hydrolysate consists of 5 mg each of cellulase and pectinase.

[0025] Furthermore, in step two, the amount of cyclohexane used is 20 mL, and the amount of ethyl acetate used each time is 20 mL.

[0026] Furthermore, in step two, the Oasis HLB solid-phase extraction column has a specification of 200 mg / 6 cc.

[0027] Furthermore, in step two, the volume ratio of methanol to acetonitrile is 1:1.

[0028] Furthermore, in step three, the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

[0029] Furthermore, the reference characteristic chromatogram generated in step four presents 10 chromatographic peaks, among which peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 correspond to the reference standards loganic acid, gentiopicrin, puerarin, astilbin, paeoniflorin, benzoylpaeoniflorin, resveratrol, rhein, succinylhexidine A, and succinylhexidine B, respectively.

[0030] Furthermore, in step four, the relative retention times of each characteristic peak and the S peak are calculated using the paeoniflorin reference peak as the S peak. The relative retention times are all within ±10% of the specified values. The specified values ​​are: 0.55 for peak 1, 0.59 for peak 2, 0.63 for peak 3, 0.69 for peak 4, 1.11 for peak 6, 1.21 for peak 7, 1.28 for peak 8, 1.50 for peak 9, and 1.71 for peak 10.

[0031] Compared with the prior art, the advantages of the present invention are as follows:

[0032] 1. More comprehensive with multiple indicators: For the first time, a characteristic chromatographic method was established to simultaneously detect 10 major active ingredients (paeoniflorin, puerarin, gentiopicrin, loganic acid, astilbene, benzoylpaeoniflorin, resveratrol, rhein, silymarin A, and silymarin B) in this compound, overcoming the limitations of controlling quality with a single ingredient and enabling a more comprehensive and objective evaluation of the overall quality of the drug;

[0033] 2. Scientific and Highly Specific Method: By optimizing sample pretreatment processes and chromatographic gradient elution conditions, interference from complex matrices is effectively eliminated, ensuring excellent separation of each component. The method is highly specific, with no negative interference, and can accurately identify target components.

[0034] 3. Stable, reliable, and highly reproducible: After systematic methodological validation, this method has demonstrated excellent performance in terms of repeatability, intermediate precision, and solution stability (RSD less than 3%), ensuring high consistency and reliability of test results under different personnel, instruments, and times, providing a solid basis for the establishment of quality standards. Attached Figure Description

[0035] The invention will now be further described with reference to the accompanying drawings;

[0036] Figure 1 This is a repeatable chromatographic overlay diagram of the present invention;

[0037] Figure 2 This is a chromatographic overlay diagram of the stability of the present invention;

[0038] Figure 3 This is a chromatogram of the reference solution for the characteristic chromatograms of this invention;

[0039] Figure 4 The chromatogram of the test sample solution is a characteristic chromatogram of the present invention. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings. The instruments and equipment used in the embodiments are as follows:

[0041] I. Instruments and Reagents

[0042] 1.1 Instruments and Equipment

[0043] .

[0044] 1.2 Test Materials

[0045] .

[0046] 1.3 Reference Standard Information

[0047] .

[0048] 1.4 Sample Information

[0049] .

[0050] The Qingjiang and Xiasuan compound of this embodiment is composed of the following components: 100 parts by weight of kudzu root, 50 parts by weight of cinnamon twig, 100 parts by weight of white peony root, 150 parts by weight of chicory, 150 parts by weight of coix seed, 100 parts by weight of smilax glabra, 100 parts by weight of dioscorea hypoglauca, 50 parts by weight of gentian root, 100 parts by weight of polygonum cuspidatum, 100 parts by weight of lysimachia christinae, and 50 parts by weight of lily bulb.

[0051] Example 1: Study on the characteristic spectral method of Shengqing Jiangsuan compound

[0052] Preparation of reference solution

[0053] Take appropriate amounts of paeoniflorin, puerarin, gentiopicrin, loganic acid, astilbene, benzoylpaeoniflorin, resveratrol, rhein, squalene A, and squalene B reference standards, accurately weigh them, and add methanol to prepare a mixed reference solution containing 60 µg paeoniflorin, 45 µg puerarin, 45 µg gentiopicrin, 30 µg loganic acid, 40 µg astilbene, 20 µg benzoylpaeoniflorin, 12 µg resveratrol, 25 µg rhein, 30 µg squalene A, and 30 µg squalene B per mL. This is the reference solution.

[0054] Preparation of the test solution: Accurately measure 10 mL of the saturation-lowering acid-reducing mixture, add 1 mol / L phosphate buffer to adjust the pH to 5.0, then add the compound enzymatic hydrolysate (containing 5 mg each of cellulase and pectinase) and hydrolyze in a water bath at 45℃ for 45 minutes. After cooling, adjust the pH to 2.5 with 10% hydrochloric acid solution, transfer to a separatory funnel, and defatt with 20 mL of cyclohexane and discard the organic phase. The aqueous phase is then extracted three times with ethyl acetate (20 mL each time). The ethyl acetate extracts are combined and evaporated under reduced pressure to dryness at 45℃. The residue is dissolved thoroughly in 10 mL of methanol to obtain a preliminary purified solution. This solution is loaded at a flow rate of 1 mL / min into an Oasis HLB solid-phase extraction column (200 mg / 6 cc) pre-activated with 5 mL of methanol and 5 mL of water. After loading, the column is first washed with 5 mL of 5% methanol aqueous solution and discarded, then pre-eluted with 5 mL of 40% methanol aqueous solution and discarded, and finally eluted with 8 mL of cyclohexane aqueous solution. The target component was eluted with an 85% methanol aqueous solution containing 0.1% formic acid, and all eluent was collected. The eluent was concentrated to near dryness by nitrogen blowing at 50°C. The residue was precisely reconstituted by ultrasonication with 1.0 mL of methanol-acetonitrile (1:1, v / v) mixed solvent. The solution was filtered through a 0.22 μm nylon microporous membrane, and the resulting filtrate was the test solution.

[0055] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result;

[0056] Determined under chromatographic conditions:

[0057] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile:methanol (volume ratio 78:22) was used as mobile phase A, and 0.1% phosphoric acid solution (containing 0.1 g sodium dodecyl sulfonate per 100 mL) was used as mobile phase B, with gradient elution as specified in the table below; column temperature was 30℃; detection wavelength was 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should not be less than 5000.

[0058] .

[0059] System Applicability

[0060] Accurately pipette 10 μL of the reference solution and repeat the injection 6 times. Calculate the retention time RSD values ​​of paeoniflorin, puerarin, gentiopicrin, loganic acid, astilbene, benzoylpaeoniflorin, resveratrol, rhein, silymarin A, and silymarin B. The theoretical plate number and retention time are shown in the table below.

[0061] Table 1 System Applicability Results

[0062] .

[0063] Table 2 System applicability retention time (min) results

[0064] .

[0065] The results show that the theoretical plate number of paeoniflorin is greater than 5000 and the RSD of retention time is less than 2.0%, indicating good system suitability.

[0066] (2) Exclusivity

[0067] Accurately pipette 10 μL each of the negative solution, reference solution, and test solution, and inject them into the liquid chromatograph for determination.

[0068] Summary: The results show that paeoniflorin, puerarin, gentiopicrin, loganic acid, astilbene, benzoylpaeoniflorin, resveratrol, rhein, silymarin A, and silymarin B in the test solution and the reference solution have the same chromatographic peaks at the corresponding retention times. The resolution of paeoniflorin, puerarin, gentiopicrin, loganic acid, astilbene, benzoylpaeoniflorin, resveratrol, rhein, silymarin A, and silymarin B from adjacent peaks is greater than 1.50, and there is no interference from the negative sample solution, indicating good method specificity.

[0069] (3) Repeatability

[0070] Accurately measure 5 mL of the saturation-lowering and acid-reducing compound, and prepare 6 test solutions according to the test solution preparation method. Inject the solutions, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the relative standard deviation. The results are as follows (see below). Figure 1 .

[0071] Table 3. Repeatability Relative Retention Time Results

[0072] .

[0073] Table 4. Repeatability of relative peak area results

[0074] .

[0075] The results show that the relative retention time RSD of each chromatographic peak is less than 2.0%, and the relative peak area RSD is less than 3.0%, indicating good repeatability.

[0076] (4) Intermediate precision

[0077] At different times, another analyst conducted the following experiments using different chromatographs.

[0078] Accurately measure 5 mL of the saturation and acid-lowering compound, take 6 portions, and prepare 6 test solutions according to the test solution preparation method. Inject the samples, determine the relative retention time and relative peak area of ​​each chromatographic peak, and calculate the relative standard deviation.

[0079] Calculate the relative retention time and relative peak area of ​​each chromatographic peak for 12 samples for repeatability and intermediate precision, and calculate the relative standard deviation.

[0080] Table 5 Results of intermediate precision relative retention time

[0081] .

[0082] Table 6 Results of intermediate precision relative peak area

[0083] .

[0084] The results show that the relative retention time RSD of each characteristic peak is less than 2.0% and the relative peak area RSD is less than 3.0%; when n=12, the relative retention time RSD of each characteristic peak is less than 2.0% and the relative peak area RSD is less than 3.0%, indicating good intermediate precision.

[0085] (5) Solution stability

[0086] Accurately measure 5 mL of the saturation-lowering and acid-reducing mixture, and prepare one test solution according to the test solution preparation method. Inject the solution at 0, 2, 4, 8, 12, and 24 hours, and determine the relative retention time and relative peak area of ​​each chromatographic peak. Calculate the relative standard deviation. See [link to relevant documentation]. Figure 2 .

[0087] Table 7 Results of relative retention time for stability

[0088] .

[0089] Table 8. Results of relative peak area for stability

[0090] .

[0091] The results show that the relative retention time RSD of each characteristic peak is less than 2.0%, the relative peak area RSD is less than 3.0%, and the solution has good stability.

[0092] (6) Characteristic spectrum determination

[0093] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile:methanol (78:22) was used as mobile phase A, and 0.1% phosphoric acid solution (containing 0.1 g sodium dodecyl sulfonate per 100 mL) was used as mobile phase B, with gradient elution as specified in the table below; column temperature was 30℃; detection wavelength was 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 5000.

[0094] .

[0095] Preparation of reference solution: Accurately weigh appropriate amounts of paeoniflorin, puerarin, gentiopicrin, loganic acid, astilbene, benzoylpaeoniflorin, resveratrol, rhein, squalene A, and squalene B reference standards, and add methanol to prepare a mixed reference solution containing 60 µg paeoniflorin, 45 µg puerarin, 45 µg gentiopicrin, 30 µg loganic acid, 40 µg astilbene, 20 µg benzoylpaeoniflorin, 12 µg resveratrol, 25 µg rhein, 30 µg squalene A, and 30 µg squalene B per mL.

[0096] Preparation of the test solution: Accurately measure 10 mL of the saturation-lowering acid-reducing mixture, add 1 mol / L phosphate buffer to adjust the pH to 5.0, then add the compound enzymatic hydrolysate (containing 5 mg each of cellulase and pectinase) and hydrolyze in a water bath at 45℃ for 45 minutes. After cooling, adjust the pH to 2.5 with 10% hydrochloric acid solution, transfer to a separatory funnel, and defatt with 20 mL of cyclohexane and discard the organic phase. The aqueous phase is then extracted three times with ethyl acetate (20 mL each time). The ethyl acetate extracts are combined and evaporated under reduced pressure to dryness at 45℃. The residue is dissolved thoroughly in 10 mL of methanol to obtain a preliminary purified solution. This solution is loaded at a flow rate of 1 mL / min into an Oasis HLB solid-phase extraction column (200 mg / 6 cc) pre-activated with 5 mL of methanol and 5 mL of water. After loading, the column is first washed with 5 mL of 5% methanol aqueous solution and discarded, then pre-eluted with 5 mL of 40% methanol aqueous solution and discarded, and finally eluted with 8 mL of cyclohexane aqueous solution. The target component was eluted with an 85% methanol aqueous solution containing 0.1% formic acid, and all eluent was collected. The eluent was concentrated to near dryness by nitrogen blowing at 50°C. The residue was precisely reconstituted by ultrasonication with 1.0 mL of methanol-acetonitrile (1:1, v / v) mixed solvent. The solution was filtered through a 0.22 μm nylon microporous membrane, and the resulting filtrate was the test solution.

[0097] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0098] In the chromatogram of the test sample (see...) Figure 4 Ten chromatographic peaks were observed. Peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 corresponded to the reference peaks of loganic acid, gentiopicrin, puerarin, astilbin, paeoniflorin, benzoylpaeoniflorin, resveratrol, rhein, squalene A, and squalene B, respectively. The paeoniflorin reference peak was designated as the S peak. The relative retention times of each characteristic peak and the S peak were calculated, and all relative retention times were within ±10% of the specified values. The specified values ​​were: 0.55 (peak 1), 0.59 (peak 2), 0.63 (peak 3), 0.69 (peak 4), 1.11 (peak 6), 1.21 (peak 7), 1.28 (peak 8), 1.50 (peak 9), and 1.71 (peak 10).

[0099] Example 2: Study on Thin-Layer Chromatography Identification Method of Shengqing Jiangsuan Mixture

[0100] (1) Using thin-layer chromatography, the kudzu root, white peony root, and Japanese knotweed were identified simultaneously:

[0101] Preparation of reference herb solutions: Take 1g of kudzu root reference herb, add 5mL of methanol, sonicate for 20 minutes, filter, and concentrate the filtrate to about 2mL on a water bath to obtain the kudzu root reference herb solution; Take 1g of white peony root reference herb, add 10mL of ethanol, warm soak for 1 hour, filter, and concentrate the filtrate to 2mL to obtain the white peony root reference herb solution; Take 0.2g of Polygonum cuspidatum reference herb, add 1mL of hydrochloric acid to moisten, add 25mL of dichloromethane, heat under reflux for 1 hour, cool, filter, evaporate the filtrate to dryness, and dissolve the residue in 1mL of anhydrous ethanol to obtain the Polygonum cuspidatum reference herb solution;

[0102] Preparation of reference solution: Take rhein and rhein methyl ether reference standards, add methanol to prepare a mixed solution containing 0.2 mg of each per 1 mL, as the reference solution;

[0103] Preparation of test solution: Take 40 mL of the acid-lowering and purifying mixture, add 2.5 mL of hydrochloric acid, and extract twice with dichloromethane, 30 mL each time. Combine the dichloromethane extracts (acid solution for later use), evaporate to dryness, and dissolve the residue in 1 mL of anhydrous ethanol to obtain test solution A; take the acid solution and extract twice with ethyl acetate, 40 mL each time. Combine the ethyl acetate extracts (acid solution for later use), evaporate to dryness, and dissolve the residue in 1 mL of methanol to obtain test solution B.

[0104] Take the acid solution and extract it twice with water-saturated n-butanol, 20 mL each time. Combine the extracts and wash them three times with water, 20 mL each time. Discard the water washings and evaporate the n-butanol solution to dryness. Dissolve the residue in 2 mL of anhydrous ethanol to obtain the C test solution.

[0105] According to the thin-layer chromatography method (General Rule 0502), take 6 μL each of the test solutions A, B, and C, 3 μL each of the reference herb solutions of kudzu root, white peony root, and polygonum cuspidatum, and 2 μL of the reference solution. Use chloroform-methanol-ethyl acetate-water-formic acid (volume ratio 33:27:12:3:0.5) as the developing solvent, develop, remove, air dry, and examine under sunlight. In the chromatogram of test solution A, two identical yellow spots appear at the corresponding positions of the chromatograms of polygonum cuspidatum and the reference solution. Examine under ultraviolet light (365 nm). In the chromatogram of test solution B, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of kudzu root reference herb. Spray with 10% sulfuric acid ethanol solution and heat at 105℃ until the spots are clearly visible. In the chromatogram of test solution C, a main spot of the same color appears at the corresponding position of the chromatogram of white peony root reference herb.

[0106] (2) Identification of Gentiana macrophylla using thin-layer chromatography:

[0107] Preparation of reference herb solution: Take 0.5g of Gentiana macrophylla reference herb, add 10mL of methanol, sonicate for 15 minutes, filter, and recover the solvent from the filtrate to 1mL to prepare the reference herb solution;

[0108] Preparation of test solution: Take 20 mL of the acid-lowering and purifying mixture, add 20 mL of methanol, filter, concentrate the filtrate to dryness, add 1 mL of methanol to dissolve, and use as the test solution;

[0109] Perform the thin-layer chromatography test (General Rule 0502). Apply 3 μL of the test solution and the reference medicinal material solution separately to the same silica gel GF254 thin-layer plate. Develop the plate using ethyl acetate-methanol-water (10:2:1) as the developing solvent. Remove the plate, air-dry it, and examine it under ultraviolet light (254 nm). In the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0110] Example 3: Study on the content determination method of Shengqing Jiangsuan compound.

[0111] Preparation of reference solution: Take appropriate amounts of paeoniflorin, puerarin and gentiopicrin reference standards, accurately weigh them, add methanol to prepare a solution containing 0.8 mg per 1 mL;

[0112] Preparation of the test solution: Accurately measure 10 mL of the saturation-lowering acid-reducing mixture, add 1 mol / L phosphate buffer to adjust the pH to 5.0, then add the compound enzymatic hydrolysate (containing 5 mg each of cellulase and pectinase) and hydrolyze in a water bath at 45℃ for 45 minutes. After cooling, adjust the pH to 2.5 with 10% hydrochloric acid solution, transfer to a separatory funnel, and defatt with 20 mL of cyclohexane and discard the organic phase. The aqueous phase is then extracted three times with ethyl acetate (20 mL each time). The ethyl acetate extracts are combined and evaporated under reduced pressure to dryness at 45℃. The residue is dissolved thoroughly in 10 mL of methanol to obtain a preliminary purified solution. This solution is loaded at a flow rate of 1 mL / min into an Oasis HLB solid-phase extraction column (200 mg / 6 cc) pre-activated with 5 mL of methanol and 5 mL of water. After loading, the column is first washed with 5 mL of 5% methanol aqueous solution and discarded, then pre-eluted with 5 mL of 40% methanol aqueous solution and discarded, and finally eluted with 8 mL of cyclohexane aqueous solution. The target component was eluted with an 85% methanol aqueous solution containing 0.1% formic acid, and all eluent was collected. The eluent was concentrated to near dryness by nitrogen blowing at 50°C. The residue was precisely reconstituted by ultrasonication with 1.0 mL of methanol-acetonitrile (1:1, v / v) mixed solvent. The solution was filtered through a 0.22 μm nylon microporous membrane, and the resulting filtrate was the test solution.

[0113] Assay: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0114] Determined under chromatographic conditions:

[0115] Chromatographic conditions: Chromatographic conditions and system suitability test

[0116] Using octadecylsilane-bonded silica gel as the packing material; acetonitrile as mobile phase A and 0.2% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in the table below; the detection wavelength was 230 nm. The theoretical plate number, calculated based on the paeoniflorin peak, should be no less than 3000.

[0117] .

[0118] (1) System applicability

[0119] Accurately pipette 10 μL of the reference solution and inject it 6 times. Calculate the retention time and peak area RSD values ​​of paeoniflorin, puerarin, and gentiopicrin. The theoretical plate number and peak area are shown in the table below.

[0120] Table 9. Theoretical plate number and asymmetry results for paeoniflorin, puerarin, and gentiopicrin.

[0121] .

[0122] Table 10 Results of the systematic applicability study of paeoniflorin, puerarin, and gentiopicrin

[0123] .

[0124] Summary: The results show that the theoretical plate numbers of paeoniflorin, puerarin, and gentiopicrin are all no less than 5000. When the same reference solution was injected six times consecutively, the retention time and peak area RSD values ​​of paeoniflorin, puerarin, and gentiopicrin were all less than 2.0%, indicating good system suitability.

[0125] (2) Exclusivity

[0126] Accurately pipette 10 μL each of the negative solution, reference solution, and test solution, and inject them into the liquid chromatograph for determination.

[0127] Summary: The results show that paeoniflorin, puerarin, and gentiopicrin in the test solution and the reference solution have the same chromatographic peaks at the corresponding retention times. The resolution of paeoniflorin, puerarin, and gentiopicrin from adjacent peaks is greater than 1.50, and there is no interference from the negative solution, indicating good method specificity.

[0128] (3) Repeatability

[0129] Take the Shengqingjiangsuan compound and prepare 6 test solutions in parallel according to the test solution preparation method. Inject them into the liquid chromatograph for determination. The results are shown in the table below.

[0130] Table 11 Results of Repeatability Testing

[0131] .

[0132] Summary: The results show that RSD < 2.0%, indicating good repeatability.

[0133] (4) Stability

[0134] Take the purifying and acid-lowering compound and prepare the test solution according to the test solution preparation method. Inject the solution into the liquid chromatograph at 0, 2, 4, 8, 12 and 24 h respectively. The results are shown in the table below.

[0135] Table 12 Stability test results

[0136] .

[0137] Summary: The results show that RSD < 2.0%, indicating good stability.

[0138] (5) Intermediate precision

[0139] Different researchers, at different times and using different instruments, prepared six parallel test solutions of the saturated and acid-lowering compound according to the test solution preparation method. The solutions were then injected into a liquid chromatograph for analysis, and the results are shown in the table below.

[0140] Table 13 Results of intermediate precision study on the contents of paeoniflorin, puerarin and gentiopicrin

[0141] .

[0142] Summary: The results show that, when different analysts used different chromatograms on different dates, the RSD of paeoniflorin, puerarin, and gentiopicrin was less than 2.0%, indicating good intermediate precision.

[0143] (6) Linearity and range

[0144] The reference solution was diluted 20-fold and injected at concentrations of 1, 3, 5, 7, 10, and 20 µL, respectively. Linear regression was performed with the mass of paeoniflorin, puerarin, and gentiopicrin as the x-axis and the peak area as the y-axis. The regression equation for paeoniflorin was: Y = 545.72X - 1.1598, R0 2 =0.9999; the regression equation for puerarin is: Y=66.522X-0.02, R 2 =1; the regression equation for gentiopicroside is: Y = 207.85X - 0.0098, R 2 =1, indicating that paeoniflorin, puerarin, and gentiopicrin showed a good linear relationship with peak area in the range of 40 μg to 800 μg. The results of the linearity investigation are shown in Table 14.

[0145] Table 14 Results of Linearity Examination

[0146] .

[0147] Summary: The results show a good linear relationship.

[0148] (7) Accuracy

[0149] Accurately measure 2.5 mL of the purifying and acid-lowering compound and place it in a 10 mL volumetric flask. Take three groups, three portions in each group, and add appropriate amounts of paeoniflorin reference standard, puerarin reference standard, and gentiopicrin reference standard to each sample. Process the samples according to the preparation method of the test solution, and determine the contents of paeoniflorin, puerarin, and gentiopicrin in each sample. Calculate the recovery rate and RSD. The results show that this method has good accuracy. The results are shown in the table below.

[0150] .

[0151] Table 15 Results of the Paeoniflorin Accuracy Test

[0152] .

[0153] Table 16 Results of the Puerarin Accuracy Test

[0154] .

[0155] Table 17 Results of the accuracy test for gentiopicroside

[0156] .

[0157] In summary, the results show that the accuracy is good.

Claims

1. A method for detecting the quality of a clear and acid mixture, characterized by, Comprise the following steps: Step one: reference solution preparation Take paeoniflorin, puerarin, gentiopicroside, loganin, astilbin, benzoylpaeoniflorin, resveratrol, rhein, pachymaran A, pachymaran B control, call, add methanol to prepare 1mL containing paeoniflorin 60ug, puerarin 45ug, gentiopicroside 45ug, loganin 30ug, astilbin 40ug, benzoylpaeoniflorin 20ug, resveratrol 12ug, rhein 25ug, pachymaran A 30ug, pachymaran B 30ug mixed solution, namely reference solution; Step two: preparation of test solution Take 10ml of shengqingjiang acid mixture, add 1mol / L phosphate buffer to adjust pH to 5.0, then add complex enzyme solution and enzyme for 45 minutes in 45℃ water bath, after cooling the enzyme solution of shengqingjiang acid mixture, adjust pH to 2.5 with 10% hydrochloric acid solution, then transfer to separatory funnel; The enzyme solution with pH adjusted to 2.5 by 10% hydrochloric acid solution was treated with cyclohexane and the organic phase was discarded, and the water phase was extracted with ethyl acetate for three times, and the ethyl acetate extract was combined and evaporated to dryness at 45℃ under reduced pressure to obtain residue, which was dissolved with 10ml of methanol to obtain the preliminary purified solution; The preliminary purified solution was loaded onto the Oasis HLB solid phase extraction column preactivated with 5ml of methanol and 5ml of water at a flow rate of 1ml / min, and then washed with 5ml of 5% methanol solution, eluted with 5ml of 40% methanol solution and discarded, and finally eluted with 8ml of 85% methanol solution containing 0.1% formic acid to collect all the eluate; The collected eluate was concentrated to near dryness under nitrogen blowing at 50℃ to obtain residue, which was ultrasonically redissolved with 1.0ml of methanol-acetonitrile mixed solvent, and the redissolved solution was filtered through 0.22um nylon microporous filter membrane to obtain the test solution; Shengqingjiang acid mixture is composed of 100 parts by weight of bauhinia, 50 parts by weight of cassia twig, 100 parts by weight of white peony root, 150 parts by weight of chicory, 150 parts by weight of yam, 100 parts by weight of smilax, 100 parts by weight of yam, 50 parts by weight of gentiana, 100 parts by weight of giant knotweed, 100 parts by weight of money grass, 50 parts by weight of lily; Step three: determination method Take 10ul of reference solution and test solution respectively, inject into liquid chromatograph, and determine; Chromatographic conditions: octadecylsilane bonded silica gel as filler; Acetonitrile: methanol (volume ratio 78:22) as mobile phase A, 0.1% phosphoric acid solution containing 0.1g of sodium dodecyl sulfate per 100ml as mobile phase B, gradient elution; Column temperature is 30℃; UV detector detection wavelength is 230nm; Theoretical plate number calculated by paeoniflorin peak should not be less than 5000; Gradient elution conditions are as follows: 0~10min, mobile phase A: B, volume ratio from 4:96 to 12:88; 10~18min, mobile phase A: B, volume ratio from 12:88 to 14:86; 18~30min, mobile phase A:B, volume ratio from 14:86 to 20:80; 30~45min, mobile phase A:B, volume ratio from 20:80 to 25:75; 45~75min, mobile phase A:B, volume ratio from 25:75 to 60:40; 75~76min, mobile phase A:B, volume ratio from 60:40 to 4:96; 76~85min, mobile phase A:B, volume ratio 4:96; Step four: generating the control characteristic map, selecting the chromatographic peaks existing in the chromatograms of different batches of Shengqing Jiangsuan Heji as common peaks, and generating the control characteristic map of Shengqing Jiangsuan Heji by using the average value calculation method.

2. The quality detection method of the ascending-sweating and descending-acid combined agent according to claim 1, characterized in that, In step two, the complex enzyme solution is 5 mg of cellulase and 5 mg of pectinase.

3. The quality detection method of the ascending-sweating and descending-acid combined agent according to claim 1, characterized in that, In step two, the amount of cyclohexane is 20 mL, and the amount of ethyl acetate is 20 mL each time.

4. The quality detection method of the ascending-sweating and descending-acid combined agent according to claim 1, characterized in that, In step two, the specification of the Oasis HLB solid-phase extraction column is 200 mg / 6 cc.

5. The method of claim 1, wherein the quality of the ascending-sweating and descending-acidic combined agent is detected. In step two, the volume ratio of methanol-acetonitrile is 1:

1.

6. The quality detection method of the ascending-sweating and descending-acid combined agent according to claim 1, characterized in that, In step three, the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm.

7. The method of claim 1, wherein the quality of the ascending-sweating and descending-acidic combined agent is determined by measuring the content of the active ingredient in the agent. In step four, the control characteristic map generated presents 10 chromatographic peaks, wherein peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, and peak 10 correspond to the reference peaks of control substances lognanc acid, gentiopicroside, paeonol, astilbin, paeoniflorin, benzoylpaeoniflorin, resveratrol, rhein, pachymaran A, and pachymaran B, respectively.

8. The method of claim 1, wherein the quality of the ascending-sweating and descending-acidic combined agent is detected. In step four, the control characteristic map generated takes the paeoniflorin reference peak as the S peak, and the relative retention time of each characteristic peak to the S peak is calculated, which is within ±10% of the specified value. The specified value is: peak 1 is 0.55, peak 2 is 0.59, peak 3 is 0.63, peak 4 is 0.69, peak 6 is 1.11, peak 7 is 1.21, peak 8 is 1.28, peak 9 is 1.50, and peak 10 is 1.71.

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