Detection method of Jianbaoling granules

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis and detection, and discloses a detection method of Jianbaoling granules. The method comprises the following steps: S1, taking an allantoin reference substance, weighing 0.5-2.0 mg of allantoin, and adding 2-5 ml of a methanol-hydrochloric acid solution for dissolving; s2, taking the content of the Jianbaoling granules, grinding, and sieving through a screen; s3, putting the content powder into a conical flask with a plug; s4, performing ultrasonic treatment on the solution; s5, uniformly stirring the mixed solution, and filtering; s6, adding methanol into residues for dissolving, and quantitatively transferring into a 10ml measuring flask; and S7, respectively taking 5-15 microliters of the reference substance solution and 5-15 microliters of the test solution. By adopting allantoin as a content determination index of functional components in the Jianbaoling granules, a set of analysis method with strong component specificity is established, accurate identification and quantification of target components are realized, and the problem of result deviation caused by improper selection of index components is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis and detection, and specifically to a detection method for Jianbaoling granules. Background Art

[0002] As a compound traditional Chinese medicine preparation, the quality control of Jianbaoling granules has long relied on the total detection of the whole extract or some conventional components. Although this method is simple to operate, its ability to target and identify key functional components in complex formulations is very weak. For example, the signals of multiple components are prone to interference with each other, resulting in the actual measured results not being able to accurately represent the true level of the pharmacodynamic components. This deviation is particularly obvious among different batches.

[0003] Currently, some detection methods use broad-spectrum indicators, such as total polysaccharides or total flavonoids. This "overall detection idea" sometimes works, but for products like Jianbaoling with complex formulations and variable components, it is very easy to have distorted judgments. Because these non-specific indicators can neither represent the core substances of the pharmacodynamic effects nor sensitively reflect the formulation consistency.

[0004] In terms of the extraction process, traditional methods mostly use conventional soaking or reflux extraction. Although this method can extract the active ingredients, the release efficiency is limited. Especially for components like allantoin with relatively poor dissolution ability, they are often not completely extracted. In many cases, the repeated fluctuations in the measurement results actually stem from incomplete extraction.

[0005] Another problem that is easily overlooked is the storage stability of the extract. In the existing technologies, there is rarely a systematic evaluation of the time stability of the extract, resulting in the situation where the components of the extract change and the concentration drifts frequently during actual detection. Sometimes the detection results are good and sometimes bad. Ultimately, it is still because the scheme is unstable and the conditions lack verification.

[0006] In addition, the chromatographic conditions often use a fixed-formula mobile phase and do not optimize the retention characteristics of the target components in complex samples. As a result, the peak separation degree is insufficient, the signal background is high, and the shape of the target peak is not ideal. In this case, even if the instrument is very precise, reliable results cannot be measured. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technologies, the present invention provides a detection method for Jianbaoling granules, which solves the problems of inaccurate identification of key components, low extraction efficiency, poor detection repeatability, and insufficient stability in the existing detection methods.

[0008] To achieve the above objectives, the present invention is realized through the following technical solutions: A detection method for Jianbaoling granules, including the following steps;

[0009] S1. Take allantoin reference substance, weigh 0.5 mg - 2.0 mg of allantoin, and then add 2 ml - 5 ml of methanol - hydrochloric acid solution to dissolve it, and prepare a reference substance solution containing 25 μg of allantoin per 1 ml of solution;

[0010] S2. Take the content of Jianbaoling granules, grind it finely and sieve it through a sieve, and take 0.5 g - 1.5 g of the content powder;

[0011] S3. Put the content powder into a stoppered conical flask, add methanol - hydrochloric acid solution and stir to mix to obtain a mixed solution, and weigh the mixed solution;

[0012] S4. Ultrasonically treat the solution, cool the treated mixed solution, weigh the mixed solution again, and use methanol - hydrochloric acid solution to make up the reduced solution volume;

[0013] S5. Stir the mixed solution evenly and then filter it, and evaporate it to dryness. Control the evaporation temperature at 40°C - 60°C to obtain a residue;

[0014] S6. Dissolve the residue in methanol and quantitatively transfer it to a 10 - ml volumetric flask, dilute it to the mark with methanol, stir evenly and then filter to obtain a test solution;

[0015] S7. Take 5 μl - 15 μl each of the reference substance solution and the test solution, inject them into a liquid chromatograph for determination.

[0016] Preferably, the ratio of methanol to hydrochloric acid is 2:1, and the hydrochloric acid concentration is 0.5 g / 100 ml.

[0017] Preferably, the mesh number range of the sieve in step S2 is 60 - 100 meshes.

[0018] Preferably, the addition amount of methanol - hydrochloric acid solution in step S3 is 20 ml - 40 ml.

[0019] Preferably, the stirring and mixing in step S3 is carried out by a vortex oscillator for 1 minute - 3 minutes.

[0020] Preferably, the ultrasonic treatment uses an ultrasonic power of 250 W - 300 W, a frequency of 40 kHz - 45 kHz, and a treatment time of 20 minutes - 40 minutes.

[0021] Preferably, the treated mixed solution in step S4 is cooled in running cold water at 15°C - 20°C for 5 minutes - 10 minutes until it reaches room temperature.

[0022] Preferably, step S7 specifically includes the following steps;

[0023] Start the liquid chromatograph and perform pre - equilibration to make the system stable and the baseline flat;

[0024] Prepare the mobile phase, degas it by ultrasound, set the flow rate to 0.5 ml / min, the detection wavelength to 191 nm - 220 nm, and the column temperature to 30 °C.

[0025] Take the reference solution and the test solution, and respectively aspirate 5 μl - 15 μl with a micro syringe.

[0026] Preferably, the chromatographic column used for the liquid chromatography analysis is a chromatographic column filled with amino-bonded silica gel, with a particle size of 5 μm and a column temperature of 25 °C - 35 °C.

[0027] Preferably, the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, mobile phase B is water, the volume ratio of acetonitrile to water is 80 - 90∶10 - 20, and the flow rate is 0.5 mL / min.

[0028] The present invention provides a detection method for Jianbaoling granules. It has the following beneficial effects:

[0029] 1. By using allantoin as the content determination index for the functional components in Jianbaoling granules, the present invention establishes a set of analytical methods with strong component specificity, realizing the accurate identification and quantification of target components. Compared with the prior art that uses non-specific components or total amount detection methods, it avoids the problem of result deviation caused by inappropriate selection of index components.

[0030] 2. By optimizing the detection process and chromatographic separation conditions, the present invention provides a quantitative detection method with good repeatability, and consistent results can still be obtained under different batches and different operators. Different from the limitations of the prior art detection methods with poor stability and susceptibility to external factors, the present invention effectively enhances the controllability and reliability of the analysis process.

[0031] 3. Through the optimization of chromatographic conditions, especially the screening of the mobile phase composition, the present invention makes the separation of allantoin in complex samples more efficient, with better peak shape symmetry and clearer detection signals. This optimization improves the selectivity and response ability of the chromatographic system to target components, providing key support for high-sensitivity and high-accuracy quantitative analysis.

[0032] 4. By adopting the method of using a methanol-hydrochloric acid dissolution system to stabilize allantoin, the present invention summarizes the mechanism of the synergistic protection of molecules by solvents and acids, achieving the technical effects of improving solubility and long-term stability. Compared with the problem of component degradation and precipitation easily caused by a single organic solvent system in the prior art, it solves the defects of poor dissolution stability and insufficient analysis accuracy of allantoin. Description of the Drawings

[0033] Figure 1 It is the flow chart of the method of the present invention. Detailed Embodiments

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] Please refer to the attached Figure 1 ;

[0036] Example 1

[0037] Prepare a reference solution: Weigh 1.0 mg of allantoin reference substance, place it in a stoppered test tube, add 4 mL of methanol-hydrochloric acid solution (the volume ratio of methanol to hydrochloric acid is 2:1, and the hydrochloric acid concentration is 0.5 g / 100 mL) to dissolve it, and prepare a reference solution containing 25 μg of allantoin in 1 mL of solution.

[0038] Prepare a test solution: Weigh 1.0 g of the content of Jianbaoling granules, grind it finely, sieve it through an 80-mesh sieve, and take the powder for use.

[0039] Put the powder into a stoppered conical flask, add 30 mL of methanol-hydrochloric acid solution, stir with a vortex oscillator for 2 minutes to form a uniformly mixed solution, and weigh the mixed solution.

[0040] Place the solution in an ultrasonic cleaner, set the ultrasonic power to 280 W, the frequency to 42 kHz, and process for 30 minutes.

[0041] After ultrasonic treatment, place the solution in flowing cold water at 15 °C to cool for 8 minutes until it reaches room temperature. Weigh again, and make up the reduced solution volume with methanol-hydrochloric acid solution.

[0042] Stir the mixed solution evenly and then filter it. Evaporate the filtrate to dryness at 50 °C to obtain a residue.

[0043] Dissolve the residue with methanol, transfer it to a 10 mL volumetric flask, add methanol to volume to the mark, shake well and filter to obtain a test solution.

[0044] Take 10 μL each of the reference solution and the test solution and inject them into a liquid chromatograph. Detection conditions: The mobile phase is acetonitrile-water (volume ratio 85:15), the flow rate is 0.5 mL / min, the detection wavelength is 220 nm, the column temperature is 30 °C, and the chromatographic column is an amino-bonded silica gel chromatographic column with a particle size of 5 μm.

[0045] Example 2

[0046] Weigh 0.5 mg of allantoin reference substance, add 2 mL of methanol-hydrochloric acid solution (volume ratio of methanol to hydrochloric acid is 2:1, hydrochloric acid concentration is 0.5 g / 100 mL) to dissolve, and prepare a reference substance solution containing 25 μg of allantoin in 1 mL of solution.

[0047] Weigh 0.5 g of the powder of the content of Jianbaoling Granules, grind it finely and sieve it through a 60-mesh sieve.

[0048] Add 20 mL of methanol-hydrochloric acid solution, vortex for 1 minute, mix well and weigh.

[0049] Ultrasonic treatment: 250 W, 40 kHz, 20 minutes.

[0050] Cool to room temperature: Cool with 15°C cold water for 5 minutes and make up for the lost volume.

[0051] Evaporate the filtrate to dryness at 40°C to obtain a residue.

[0052] Dissolve the residue with methanol, make up the volume to 10 mL, shake well and filter to obtain the test solution.

[0053] The injection volume is 5 μL. The liquid chromatography detection conditions are acetonitrile-water (volume ratio 80:20), flow rate 0.5 mL / min, column temperature 25°C, wavelength 220 nm, and the chromatographic column is an amino-bonded silica gel column with a particle size of 5 μm.

[0054] Example 3

[0055] Weigh 2.0 mg of allantoin reference substance, add 5 mL of methanol-hydrochloric acid solution (volume ratio of methanol to hydrochloric acid is 2:1, hydrochloric acid concentration is 0.5 g / 100 mL), and prepare an allantoin solution containing 25 μg in 1 mL of solution.

[0056] Take 1.5 g of the powder of the content of Jianbaoling Granules and sieve it through a 100-mesh sieve.

[0057] Add 40 mL of methanol-hydrochloric acid solution, stir with a vortex oscillator for 3 minutes to form a mixed solution and weigh.

[0058] Perform ultrasonic treatment: power 300 W, frequency 45 kHz, treat for 40 minutes.

[0059] Cooling conditions: Cool in flowing 20°C cold water for 10 minutes to room temperature and make up for the weight loss.

[0060] Evaporate the filtrate to dryness at 60°C to obtain a residue.

[0061] Dissolve the residue with methanol, make up the volume to 10 mL, filter to obtain the test solution.

[0062] Chromatographic detection: Take 15 μL of the sample. The mobile phase is acetonitrile - water (volume ratio 90:10), the flow rate is 0.5 mL / min, the column temperature is 35 °C, the wavelength is 191 nm, and the chromatographic column is amino - bonded silica gel with a particle size of 5 μm.

[0063] Comparative Example 1: Compared with Example 1, the difference is that allantoin is not used as a detection index, and the rest are the same.

[0064] Comparative Example 2: Compared with Example 1, the difference is that the allantoin reference substance is not dissolved in the methanol - hydrochloric acid system, and the rest are the same.

[0065] Comparative Example 3: Compared with Example 1, the difference is that ultrasonic treatment is not carried out, and the ultrasonic - assisted extraction step is omitted, and the rest are the same.

[0066] Comparative Example 4: Compared with Example 1, the difference is that the mobile phase of liquid chromatography is changed to a methanol - water mixture, and the rest are the same.

[0067] Comparative Experiment 1: The influence of whether to use an allantoin reference substance on the detection results

[0068] Experimental purpose

[0069] Verify whether not setting allantoin as a detection index (without a reference substance) affects the recognition and quantitative analysis ability of allantoin components in Jianbaoling granules.

[0070] Experimental groups

[0071] Experimental group (Example 1): Set an allantoin reference substance and inject samples for detection according to the standard method.

[0072] Control group (Comparative Example 1): Do not set an allantoin reference substance, and only prepare test samples for detection.

[0073] Prepare test sample solutions according to the methods of Example 1 and Comparative Example 1 respectively.

[0074] Pre - heat the liquid chromatograph for 30 minutes, prepare the mobile phase acetonitrile - water (85:15), the flow rate is 0.5 mL / min, the detection wavelength is 220 nm, the column temperature is 30 °C, and the chromatographic column is an amino - bonded silica gel column (5 μm).

[0075] First inject 10 μL of the allantoin reference substance solution into the Example 1 group, record the retention time and peak area, and establish a standard curve.

[0076] Inject 10 μL of the test sample solutions of the Example 1 group and the Comparative Example 1 group respectively, and observe whether the position, peak area, and peak shape of the allantoin peak are obvious and whether they can correspond to the standard peak.

[0077] Three batches of samples were tested in each group, and each batch was injected twice. The mean value was taken and the RSD (%) was calculated as the repeatability index.

[0078] Table 1: Experimental data on the effect of setting allantoin reference substance on test results:

[0079]

[0080] Summarize;

[0081] In this experiment, a standard curve was established by setting up an allantoin reference substance, so that the chromatographic analysis has a clear quantitative basis, and the characteristic peaks of the allantoin component in the Jianbaoling granules are effectively identified. Allantoin is a key indicator component in the detection system of the present invention. It has strong ultraviolet absorption ability, shows a good response at the selected wavelength, and has a stable retention time. It can be significantly enriched after methanol-hydrochloric acid extraction and ultrasonic treatment, and therefore can accurately reflect the level of effective components in the preparation. In contrast, the detection system without a reference substance cannot provide a standard comparison basis. Although a weak peak signal can be seen in the chromatogram, its attribution cannot be confirmed, and the response value fluctuates greatly, lacking data traceability.

[0082] The chromatographic detection method established by the present invention is designed around the physicochemical properties of allantoin under specific conditions. Since allantoin contains carboxyl and amide structures in its molecular structure, it has a strong polarity and can form a clear separation effect in an acetonitrile-water system, and has good retention performance on an amino bonded phase column. The setting of the standard solution not only ensures the identifiability of the detection peak, but also ensures the scientific nature of the content determination through a linear relationship. In the absence of a standard solution, even if the same extraction process and chromatographic conditions are used, it is difficult to effectively resolve the target component from a complex matrix, resulting in the method losing reliability in actual quality control.

[0083] The experimental results fully demonstrate that establishing a standard control system is not only the foundation of quantitative analysis but also a key link in demonstrating the scientific and practical nature of this invention. By introducing the allantoin reference substance as an analytical anchor, the entire detection system operates stably, achieving highly targeted and accurate quantitative control capabilities.

[0084] Comparative Experiment 2: Effect of the Dissolution Method of the Reference Substance on the Test Results

[0085] Purpose of the experiment

[0086] The effect of dissolving the reference substance allantoin in different solvent systems (methanol-hydrochloric acid vs. pure methanol) on the test results was verified, and its impact on the linearity of the standard curve, peak shape and accuracy of content determination was evaluated.

[0087] Experimental groups

[0088] Experimental group (Example 1): Allantoin reference substance was dissolved in methanol - hydrochloric acid solution (volume ratio 2:1, hydrochloric acid concentration 0.5 g / 100 mL).

[0089] Control group (Comparative Example 2): Allantoin reference substance was dissolved in pure methanol.

[0090] Experimental procedures

[0091] Preparation of reference substance solution:

[0092] Experimental group: Weigh the allantoin reference substance and dissolve it in methanol - hydrochloric acid solution to prepare a standard solution with a certain concentration.

[0093] Control group: Weigh the same amount of allantoin reference substance and dissolve it in pure methanol to prepare a standard solution with the same concentration.

[0094] Preparation of test sample solution: Prepare the test sample solution according to the method of Example 1.

[0095] Setting of liquid chromatography conditions:

[0096] Chromatographic column: Amino - bonded silica gel column (5 μm).

[0097] Mobile phase: Acetonitrile - water (volume ratio 85:15).

[0098] Flow rate: 0.5 mL / min.

[0099] Detection wavelength: 220 nm.

[0100] Column temperature: 30 °C.

[0101] Injection volume: 10 μL.

[0102] Drawing of standard curve:

[0103] Use the reference substance solutions of the experimental group and the control group respectively to prepare standard solutions with different concentration gradients.

[0104] Inject for determination, draw the standard curve, and calculate the linear correlation coefficient (R 2 ).

[0105] Sample determination:

[0106] Use their respective standard curves to determine the content of allantoin in the test sample.

[0107] Detect 3 batches of samples for each group, inject 2 times for each batch, take the average value and calculate RSD (%) as the repeatability index.

[0108] Table 2: Experimental data on the influence of the dissolution method of the reference substance on the detection results

[0109]

[0110] Summary:

[0111] In the present invention, allantoin is selected as the key detection index. Its structure contains amide and carboxyl functional groups and has a certain polarity. In a pure methanol system, due to the insufficient polarity of the solvent, the solubility of allantoin is limited, and it is very easy to crystallize out or form an incompletely dissolved state, resulting in unstable reference substance concentration, which in turn affects the linear relationship and signal response of chromatographic detection. In contrast, the methanol-hydrochloric acid mixed system can enhance the dissociation degree of allantoin molecules through an acidic environment while increasing the polarity, which helps to form a clear and stable standard solution and provides a basis for subsequent quantitative analysis.

[0112] From the perspective of the detection mechanism, chromatographic quantification depends on the accuracy of the standard curve, and the construction of the standard curve highly depends on the dissolution state and concentration stability of the reference substance solution. If the reference substance fails to dissolve sufficiently, it will lead to fluctuations in peak area, low response value, and even abnormal peak shapes such as tailing and splitting. In the experiment, it was observed that the peak shape of the reference substance dissolved in pure methanol became worse and the repeatability decreased, which was exactly due to the mismatch between the solvent system and the physicochemical properties of the target component, resulting in the accumulation of errors in the analysis process.

[0113] The present invention uses methanol-hydrochloric acid as the solvent system, precisely considering the mechanism that allantoin is more likely to form a stable solution in this environment, so as to ensure the controllable behavior and stable response of the reference substance during injection detection.

[0114] Comparative Experiment 3: Influence of Ultrasonic-Assisted Extraction on the Extraction Efficiency of Target Components

[0115] Experimental Purpose

[0116] To explore the role of ultrasonic treatment in the sample extraction process and verify its influence on the extraction efficiency and detection stability of allantoin.

[0117] Experimental Groups

[0118] Experimental Group (Example 1): Add an ultrasonic treatment step (280 W, 42 kHz, 30 minutes) after sample soaking.

[0119] Control Group (Comparative Example 3): Omit ultrasonic treatment, and the sample is only extracted by natural static placement.

[0120] Experimental Steps

[0121] Preparation of Test Solution: Each group soaks in a methanol-hydrochloric acid mixed solution according to the original process; the experimental group is then placed in an ultrasonic cleaner for treatment, while the control group is not treated.

[0122] Post-Extraction Treatment: The solutions are cooled, evaporated to dryness, and fixed volume to 10 mL respectively to obtain the test solution.

[0123] Liquid Chromatography Conditions Setting:

[0124] Chromatographic column: Amino-bonded silica gel column (5μm)

[0125] Mobile phase: Acetonitrile - water (85:15)

[0126] Flow rate: 0.5 mL / min

[0127] Detection wavelength: 220 nm

[0128] Column temperature: 30 °C

[0129] Injection volume: 10 μL

[0130] Detection process: Each group detects 3 batches of samples, injects samples 2 times for each batch, and observes the retention time, peak area, content, and repeatability of allantoin peaks

[0131] Table 3: Experimental data on the influence of ultrasonic treatment on detection results

[0132] Group Peak area (mAU·s) Content (mg / g) Peak shape evaluation RSD (%) Example 1 2033.7 0.104 Good symmetry 2.5 Example 1 2082.5 0.107 Good symmetry Example 1 2012.1 0.103 Good symmetry Comparative Example 3 1271.6 0.066 Low fluctuation 6.1 Comparative Example 3 1345.8 0.07 Weak peak Comparative Example 3 1189.2 0.062 Incomplete

[0133] Summary:

[0134] During the extraction process of the target component, the dense structure or local embedding of the sample often restricts the penetration and effective contact of the solvent. Especially in short-time extraction, it is likely to lead to insufficient release of the target component, thereby affecting the accuracy and reproducibility of the detection results. When ultrasonic waves propagate in a liquid, they can form a "cavitation effect", that is, the formation and collapse of tiny bubbles can instantaneously generate high pressure and high temperature locally, effectively destroying the plant cell structure and improving the release efficiency of solutes. Therefore, introducing ultrasonic treatment in the experiment is based on the physical extraction mechanism, optimizing the interaction mode between the solvent and the sample, and thus more efficiently releasing target components such as allantoin.

[0135] From the perspective of chromatographic detection, the difference in extraction efficiency directly affects the peak area size and repeatability performance. If the extraction of the target component is insufficient and the actual injection volume is low, it is likely to lead to unstable signal response, and even phenomena such as peak shape drift or weak peaks. In the experiment, it was observed that the sample group without ultrasonic treatment showed a significant reduction in peak area, a weakening of the peak shape, and large batch-to-batch fluctuations, indicating incomplete extraction and poor detection repeatability. While the sample group assisted by ultrasound not only had stable content, but also had sharp peak shapes and good reproducibility, reflecting the decisive role of the extraction process on the subsequent analysis quality.

[0136] In the present invention, the improvement of the extraction method is based on the overall consideration of the physical and chemical properties of the target component and the sample structure. By introducing ultrasonic physical enhancement means, the extraction effect is significantly improved, enabling the target component to enter the analysis system more completely and uniformly, and improving the reliability of the analysis process.

[0137] Comparative Experiment 4: Influence of Mobile Phase Composition on the Chromatographic Behavior of Allantoin

[0138] Experimental Purpose

[0139] Investigate the influence of mobile phase selection (acetonitrile - water vs methanol - water) on the chromatographic retention behavior, separation effect, and peak shape performance of allantoin, and verify its impact on the accuracy and reliability of the analysis results.

[0140] Experimental Group

[0141] Experimental Group (Example 1): Use acetonitrile - water (85:15, v / v) as the mobile phase.

[0142] Control Group (Comparative Example 4): Use methanol - water (85:15, v / v) as the mobile phase.

[0143] Experimental Procedure

[0144] Preparation of Test Solution: Use the same batch of test solution for each group to ensure sample consistency.

[0145] Setting of Chromatographic Conditions (kept consistent except for the mobile phase):

[0146] Chromatographic Column: Amino - bonded silica gel column (5μm)

[0147] Flow Rate: 0.5 mL / min

[0148] Detection Wavelength: 220 nm

[0149] Column Temperature: 30 °C

[0150] Injection Volume: 10 μL

[0151] Detection Process: Inject the same test sample with two mobile phase systems respectively, and record the retention time, peak shape, resolution of the allantoin peak, and the presence of interference peaks or overlapping phenomena.

[0152] Table 4: Comparison of Allantoin Chromatographic Behavior under Different Mobile Phase Systems

[0153]

[0154]

[0155] Summary;

[0156] The core mechanism of chromatographic separation lies in the partitioning behavior of target compounds between the stationary phase and the mobile phase. Its retention time, peak shape, and resolution are jointly affected by factors such as the polarity of the mobile phase, solvent strength, and intermolecular interactions. Allantoin, as a small molecule with moderate polarity and stable structure, is particularly sensitive to the mobile phase in a reverse-phase chromatography system. Acetonitrile has lower viscosity and higher solvent strength compared to methanol, which can reduce the system backpressure, reduce band broadening, and improve the migration efficiency of solutes in the column, thereby improving the peak shape performance and enhancing the separation ability between components.

[0157] Judging from the experimental results, when methanol is used as the mobile phase, the retention time of allantoin is significantly advanced, and at the same time, phenomena such as peak shape diffusion, tailing, and adjacent peak overlap occur. This reflects the lack of sufficient selectivity between the mobile phase and the target component, resulting in a decline in the separation effect, interference component signals mixing into the analysis window, and directly threatening the accuracy of qualitative judgment and the reliability of quantitative data. After using the acetonitrile system, not only is the retention of the target peak more stable, but also the peak shape is clear and the resolution is significantly improved, demonstrating better elution kinetic conditions and effectively avoiding interference from impurity peaks.

[0158] The present invention optimizes the composition of the mobile phase to highly match the chromatographic behavior with the characteristics of the target component, realizing the effective identification and separation of allantoin and ensuring the scientificity and repeatability of the detection data.

[0159] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A detection method for Jianbaoling granules, characterized in that, It includes the following steps; S1. Take allantoin reference substance, weigh 0.5 mg - 2.0 mg of allantoin, and then add 2 ml - 5 ml of methanol - hydrochloric acid solution to dissolve it, and prepare a reference substance solution containing 25 μg of allantoin per 1 ml of solution; S2. Take the content of Jianbaoling granules, grind it finely and sieve it through a sieve, and take 0.5 g - 1.5 g of the content powder; S3. Put the content powder into a stoppered conical flask, add methanol - hydrochloric acid solution and stir to mix to obtain a mixed solution, and weigh the mixed solution; S4. Ultrasonically treat the solution, cool the treated mixed solution, weigh the mixed solution again, and make up the reduced solution volume with methanol - hydrochloric acid solution; S5. Stir the mixed solution evenly and then filter it, and evaporate it to dryness. The evaporation temperature is controlled at 40°C - 60°C to obtain a residue; S6. Dissolve the residue with methanol and quantitatively transfer it to a 10 - ml volumetric flask, dilute it to the mark with methanol, stir evenly and then filter to obtain a test solution; S7. Take 5 μl - 15 μl each of the reference substance solution and the test solution, and inject them into a liquid chromatograph for determination.

2. The detection method of Jianbaoling granules according to claim 1, characterized in that The ratio of methanol to hydrochloric acid is 2∶1, and the hydrochloric acid concentration is 0.5 g / 100 ml.

3. The detection method of Jianbaoling Granules according to claim 1, characterized in that The mesh number range of the sieve in step S2 is 60 - 100 meshes.

4. The detection method of Jianbaoling Granules according to claim 1, characterized in that, The addition amount of methanol - hydrochloric acid solution in step S3 is 20 ml - 40 ml.

5. The detection method of Jianbaoling Granules according to claim 1, characterized in that, In step S3, the stirring and mixing is carried out by a vortex oscillator for 1 minute - 3 minutes.

6. The detection method of Jianbaoling Granules according to claim 1, characterized in that, The ultrasonic treatment is carried out with an ultrasonic power of 250 W - 300 W, a frequency of 40 kHz - 45 kHz, and a treatment time of 20 minutes - 40 minutes.

7. The detection method of Jianbaoling granules according to claim 1, characterized in that, In step S4, the treated mixed solution is cooled in flowing cold water at 15°C - 20°C for 5 minutes - 10 minutes until it reaches room temperature.

8. The detection method of a Jianbaoling granule according to claim 1, characterized in that, Step S7 specifically includes the following steps; Enable the liquid chromatograph and perform pre - equilibration to make the system stable and the baseline flat; Configure the mobile phase, use ultrasonic degassing, set the flow rate to 0.5 ml / min, the detection wavelength to 191 nm - 220 nm, and the column temperature to 30°C. Take the reference substance solution and the test solution, and respectively suck 5 μl - 15 μl with a micro - syringe; Inject the reference substance solution into the liquid chromatograph and record its retention time and peak area data; Inject the test solution into the liquid chromatograph in sequence, observe the position of the allantoin peak, and record the peak area; Calculate the content of allantoin in the test sample by the external standard method.

9. The detection method of Jianbaoling Granules according to claim 8, characterized in that, The chromatographic column used for the liquid chromatographic analysis is a chromatographic column filled with amino - bonded silica gel, with a particle size of 5 μm and a column temperature of 25°C - 35°C.

10. The detection method of Jianbaoling Granules according to claim 8, characterized in that, The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is acetonitrile, mobile phase B is water, and the volume ratio of acetonitrile to water is 80 - 90∶10 - 20, and the flow rate is 0.5 mL / min.

Images