Construction method of compound xianghua granules fingerprint spectrum

Abstract

The application discloses a kind of compound Xianghua granules fingerprint's construction method, comprising the following steps: preparation of reference solution: p-coumaric acid, ferulic acid standard in methanol is dissolved;Preparation of test solution: different batches of compound Xianghua granules are dissolved in ultrapure water respectively, and post-processing is carried out;Respectively inject high performance liquid chromatography and detect, obtain the fingerprint of reference solution and the fingerprint of each batch sample;Using traditional Chinese medicine fingerprint similarity evaluation system analyzes the fingerprint of each batch sample, generates the control fingerprint of compound Xianghua granules.The method provided by the application can better reflect the multiple index component information of compound Xianghua granules.The fingerprint characteristic peak baseline of the application is smooth, and the sensitivity and separation degree are better, while the operation is quick and reliable, the cost is lower, can effectively, stably and quickly comprehensively monitor product quality, ensure the stability and quality controllability of product.

Description

A method for constructing the fingerprint spectrum of compound fragrant flower granules Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis technology, and in particular to a method for constructing a fingerprint spectrum of compound fragrant flower granules. Background Technology

[0002] Chronic insomnia refers to a long-term deficiency in the quality or quantity of sleep, causing impairment of behavioral and psychological function, and is generally accompanied by other related clinical symptoms. Chronic insomnia is one of the most common sleep disorders in clinical practice. Its etiology is complex, affecting the physical and mental health, cognitive function, memory, creative abilities, and social activities of the general population. Specific manifestations include difficulty falling asleep, difficulty maintaining sleep, and decreased sleep quality. Traditional Chinese medicine (TCM) generally diagnoses the pathogenesis of insomnia as "Yang failing to enter Yin," meaning that "Wei Qi (defensive Qi) cannot enter Yin and often remains in Yang. When it remains in Yang, Yang Qi is abundant, and when Yang Qi is abundant, Yang Qiao (the yang meridian) is excessive; when it cannot enter Yin, Yin Qi is deficient, hence the inability to close the eyes." Traditionally, treatment follows principles such as "treating from the perspective of the heart" and "treating from the perspective of the liver."

[0003] Compound Xianghua Granules are a traditional Chinese medicine compound preparation derived from the hospital's in-house preparation, Luohua Anshen Oral Liquid, with modifications based on clinical experience. It consists of two herbs: peanut branches and leaves, and vinegar-processed Cyperus rotundus. Clinical application shows that Compound Xianghua Granules have a higher overall effective rate than Luohua Anshen Oral Liquid in treating simple insomnia and cases with insomnia as the primary symptom and multiple complications, indicating good development potential. The characteristics of TCM syndrome differentiation and treatment dictate that the formulation needs to be adjusted according to the changes in the disease to best adapt to complex pathological syndromes. Therefore, research on Compound Xianghua Granules, this in-house preparation with "modification according to syndrome," is of practical significance. However, due to the complexity of TCM components, research on single or only a few effective components does not conform to the characteristics of multi-component, multi-target treatment in TCM, nor can it achieve overall monitoring of the finished product quality, let alone ensure the reproducibility of efficacy. Therefore, establishing a fingerprinting method that includes information on the components of a multi-herb compound preparation using modern pharmaceutical and analytical techniques is of great significance, providing a solid foundation for non-clinical and clinical experimental research of the preparation. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for constructing the fingerprint spectrum of compound fragrant flower granules.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for constructing the fingerprint spectrum of compound fragrant flower granules is provided, including the following steps:

[0007] Step 1, Preparation of reference solution: Dissolve p-coumaric acid and ferulic acid standards in methanol to obtain the solution;

[0008] Step 2, prepare the test solution: Dissolve different batches of compound fragrant flower granules in ultrapure water and perform post-processing to obtain the solution;

[0009] Step 3: Inject the reference solution and the test solution into a high-performance liquid chromatograph for detection, and obtain the fingerprint chromatogram of the reference solution and the fingerprint chromatogram of each batch of samples, respectively.

[0010] Step 4: The fingerprint spectrum of each batch of samples is analyzed using a traditional Chinese medicine fingerprint spectrum similarity evaluation system to generate the finished product fingerprint spectrum of Compound Fragrant Flower Granules.

[0011] In step three, the conditions for high-performance liquid chromatography are as follows:

[0012] The chromatographic column length is 100mm-200mm; the column inner diameter is 3mm-6mm; the column packing material is octadecylsilane-bonded silica gel with a particle size of 3.0μm-6.0μm; the detection wavelength is 300nm-320nm; the flow rate is 0.75-0.85ml / min; the detection temperature is 27-33℃; the injection volume is 5-15μl; the theoretical plate number, calculated based on the ferulic acid peak, should not be less than 3000; the elution method is gradient elution; mobile phase A is acetonitrile, and mobile phase B is a 0.1% (v / v) aqueous solution of phosphoric acid; the gradient elution program includes:

[0013] 0 min, 5% acetonitrile, 95% 0.1% phosphoric acid aqueous solution;

[0014] 5 min, 5% acetonitrile, 95% 0.1% phosphoric acid aqueous solution;

[0015] 12 min, acetonitrile 12%, 0.1% phosphoric acid aqueous solution 88%;

[0016] 20 min, acetonitrile 12%, 0.1% phosphoric acid aqueous solution 88%;

[0017] 40 min, acetonitrile 30%, 0.1% phosphoric acid aqueous solution 70%;

[0018] 44 min, 80% acetonitrile, 20% 0.1% phosphoric acid aqueous solution;

[0019] 46 min, acetonitrile 80%, 0.1% phosphoric acid aqueous solution 20%;

[0020] 46.1 min, acetonitrile 5%, 0.1% phosphoric acid aqueous solution 95%;

[0021] 48 min, 5% acetonitrile, 95% 0.1% phosphoric acid aqueous solution.

[0022] Furthermore, in the reference solution, the mass-to-volume ratio of p-coumaric acid and ferulic acid is 5 μg / mL to 20 μg / mL.

[0023] Furthermore, in the test solution, the ratio of compound fragrant flower granules to ultrapure water is (0.5-2)g:(10-20)mL; the post-processing specifically involves: first ultrasonic treatment, and then filtration treatment.

[0024] Furthermore, the ultrasonic treatment has a power of 250W, a frequency of 40kHz, and an ultrasonic time of 30min.

[0025] Furthermore, the fingerprint spectrum of the reference solution includes: peak 3 corresponding to the coumaric acid and peak 4 corresponding to the ferulic acid.

[0026] Furthermore, the comparative fingerprint spectrum of the compound fragrant flower granules includes 5 characteristic peaks, of which peak 3 is the characteristic peak of p-coumaric acid and peak 4 is the characteristic peak of ferulic acid.

[0027] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0028] This invention provides a novel method for quality control of compound fragrant flower granules, which can better reflect the information of multiple indicative components of the compound fragrant flower granules. The fingerprint spectrum of this invention has stable baseline peaks, good sensitivity and separation, and is quick, reliable, and low-cost. It can effectively, stably, and quickly monitor product quality comprehensively, ensuring product stability and quality controllability. Attached Figure Description

[0029] Figure 1 shows the specificity test results at 310 nm in the embodiments of the present invention. In this figure, A is the chromatogram of the test solution of compound Xianghua granules (coumaric acid corresponds to peak 3 and ferulic acid corresponds to peak 4); B is the chromatogram of the reference medicinal material solution of peanut branches and leaves; C is the chromatogram of the mixed reference solution of coumaric acid and ferulic acid; and D is the chromatogram of the reference medicinal material solution of vinegar-processed Cyperus rotundus.

[0030] Figure 2 is a superimposed HPLC characteristic fingerprint spectrum of three batches of finished products at 310 nm in the embodiment of the present invention (peak 3 corresponds to coumaric acid and peak 4 corresponds to ferulic acid).

[0031] Figure 3 is a comparative fingerprint spectrum of the compound fragrant flower particles at 310 nm in the embodiment of the present invention. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0033] 1. Instruments and Reagents

[0034] Instruments: Agilent high-performance liquid chromatograph (Agilent Technologies, Agilent 1290), InfinityLab Poroshell 120 EC-C18 (4.6×100mm, 5μm) column, 0.001% balance (Mettler-Toledo, ME104), 0.1% balance (Mettler-Toledo, XPR2), CNC ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd., KQ-300DB), pure water system (Shanghai Laike Instrument Co., Ltd., OKP-S050D), benchtop high-speed refrigerated centrifuge (Shanghai Lishen Scientific Instrument Co., Ltd., Neofuuge 15R).

[0035] Reagents: Chromatographic grade acetonitrile (Shanghai Xingke High Purity Solvent Co., Ltd.), chromatographic grade methanol (Shanghai Xingke High Purity Solvent Co., Ltd.), chromatographic grade phosphoric acid (Shanghai Xingke High Purity Solvent Co., Ltd.), and ultrapure water was prepared in the laboratory using a pure water system.

[0036] Test drugs: p-coumaric acid (content 97.2%, Shanghai Shidande Standard Technical Service Co., Ltd., batch number: 7400-08-0); ferulic acid (content 97.2%, China National Institutes for Food and Drug Control, batch number: 110773-202316); peanut branches and leaves (Sichuan Xinhehua Traditional Chinese Medicine Pieces Co., Ltd., batch number: 20201026-4); vinegar-processed Cyperus rotundus (Shanghai Wanshicheng Co., Ltd., batch number: 2010105); compound fragrant flower granules (Shanghai Traditional Chinese Medicine Pharmaceutical Technology Co., Ltd., batch numbers 2201, 2202, 2203).

[0037] 2. Chromatographic conditions and system suitability conditions

[0038] The column was packed with octadecylsilane-bonded silica gel (100 mm column length, 4.6 mm inner diameter, 5 µm particle size); acetonitrile was used as mobile phase A, and 0.1% v / v phosphoric acid solution was used as mobile phase B. The detection wavelength was 310 nm, the flow rate was 0.8 mL / min, the detection temperature was 30 °C, the injection volume was 10 μL, and the theoretical plate number, calculated based on the ferulic acid peak, should not be less than 3000. Gradient elution was performed according to the specifications in Table 1.

[0039] Table 1

[0040] Time (minutes) Mobile Phase A (%) Mobile Phase B (%) 0 595 5595 12 12 88 20 12 88 40 30 70 44 80 20 46 80 20 46.15 95 48 595 0595 surface

[0041] 3. Sample Preparation

[0042] Test solution: Take 0.5g of this product, add 10ml of water, and sonicate (power 250W, frequency 40KHz) for 30min. Filter and collect the filtrate to obtain the test solution.

[0043] Reference solution: Accurately weigh appropriate amounts of ferulic acid and p-coumaric acid reference standards, dissolve them in methanol, and prepare a mixed solution containing 10 μg of each reference standard per 1 ml.

[0044] 4. Validation of HPLC characteristic chromatographic method

[0045] 4.1 Specificity Examination

[0046] Take appropriate amounts of the finished product (batch number 2201), coumaric acid, and ferulic acid reference standards, and prepare the test solution and reference standard mixed solution of Compound Xianghua Granules according to "3. Sample Preparation". Separately, take appropriate amounts of peanut branches and leaves and vinegar-processed Cyperus rotundus medicinal materials, and prepare reference medicinal material solutions according to "3. Sample Preparation". Perform HPLC analysis according to "2. Chromatographic Conditions and System Suitability Conditions", record the chromatograms, and the results are shown in Figure 1.

[0047] Experimental results: At a detection wavelength of 310 nm, the test solution showed 5 characteristic peaks, which were the same as the characteristic peaks in the chromatograms of the reference solution and the reference medicinal material at the same wavelength and retention time, and there was no negative interference, indicating that the method has good specificity.

[0048] 4.2 Precision Examination

[0049] Take an appropriate amount of the finished product (batch number 2201), prepare the test solution according to "3. Sample preparation", and analyze it according to "2. Chromatographic conditions and system suitability conditions". Inject the same test solution 6 times consecutively. Using coumaric acid as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak at 310 nm, and calculate the RSD value. The results are shown in Table 2-3.

[0050] Table 2. Results of Relative Retention Time (RRT) in Precision Study

[0051] Peak Name Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Precision -1 0.580 0.820 1.000 1.218 1.525 Precision -2 0.580 0.819 1.000 1.218 1.523 Precision -3 0.581 0.819 1.000 1.217 1.521 Precision -4 0.581 0.819 1.000 1.217 1.521 Precision -5 0.581 0.819 1.000 1.217 1.521 Precision -6 0.580 0.819 1.000 1.217 1.522 RSD% 0.08 0.05 0.00 0.04 0.10 surface

[0052] Table 3. Results of Relative Peak Area (RPA) for Precision Testing

[0053] Peak Name Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Precision -1 3.364 0.525 1.000 0.528 1.620 Precision -2 3.384 0.523 1.000 0.536 1.634 Precision -3 3.384 0.526 1.000 0.534 1.635 Precision -4 3.384 0.525 1.000 0.533 1.631 Precision -5 3.399 0.526 1.000 0.536 1.636 Precision -6 3.397 0.530 1.000 0.540 1.632 RSD% 0.37 0.46 0.00 0.77 0.37 surface

[0054] When the same sample solution was injected six times consecutively, the relative retention time RSD value with p-coumaric acid as the reference peak was in the range of 0.00-0.10%, and the relative peak area RSD value was in the range of 0.00-0.77%, both less than 5.0%, indicating that the instrument precision was good.

[0055] 4.3 Repeatability Test

[0056] Take an appropriate amount of the finished product (batch number 2201) and prepare 6 test solutions in parallel according to "3. Sample Preparation". Analyze according to "2. Chromatographic Conditions and System Suitability Conditions". Using coumaric acid as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak at 310 nm, and calculate the RSD value. The experimental results are shown in Table 4-5.

[0057] Table 4. Repeatability Relative Retention Time (RRT) Results

[0058] Peak Name Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Repeatability -1 0.580 0.819 1.000 1.217 1.522 Repeatability -2 0.580 0.819 1.000 1.216 1.522 Repeatability -3 0.581 0.819 1.000 1.217 1.522 Repeatability -4 0.580 0.819 1.000 1.217 1.522 Repeatability -5 0.580 0.820 1.000 1.218 1.523 Repeatability -6 0.580 0.820 1.000 1.218 1.524 RSD% 0.05 0.03 0.00 0.05 0.05 surface

[0059] Table 5. Repeatability Relative Peak Area (RPA) Results

[0060] Peak Name Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 Repeatability -1 3.429 0.523 1.000 0.532 1.741 Repeatability -2 3.454 0.521 1.000 0.535 1.742 Repeatability -3 3.457 0.517 1.000 0.531 1.734 Repeatability -4 3.456 0.517 1.000 0.532 1.734 Repeatability -5 3.470 0.522 1.000 0.532 1.740 Repeatability -6 3.366 0.519 1.000 0.536 1.687 RSD% 1.10 0.54 0.00 0.34 1.22 surface

[0061] Six parallel test solutions were prepared using the same instrument and equipment, with p-coumaric acid as the reference peak. The relative retention time RSD values ​​at each detection wavelength ranged from 0.00 to 0.05%, and the relative peak area RSD values ​​ranged from 0.00 to 1.22%, all less than 5.0%, indicating good sample repeatability.

[0062] 4.4 Stability Study of the Test Solution

[0063] Take an appropriate amount of the finished formulation (batch number 2201) and prepare the test solution according to "3. Sample Preparation". Analyze according to "2. Chromatographic Conditions and System Suitability Conditions". Inject the sample at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h. Using coumaric acid as the reference peak S, calculate the relative retention time and relative peak area at 310nm. The experimental results are shown in Tables 6-7.

[0064] Table 6. Relative Retention Time (RRT) Results of Test Solution Stability

[0065] Peak Name Peak 1 Peak 2 Peak 3 (S) Peak 4 50h 0.574 0.820 1.000 1.216 1.5222h 0.576 0.818 1.000 1.215 1.5194h 0.576 0.819 1.000 1.217 1.5186h 0.576 0.819 1.000 1.217 1.5198h 0.57 60.8191.0001.2161.52310h0.5750.8181.0001.2171.52212h0.5760.8181. 0001.2161.51924h0.5760.8191.0001.2151.520RSD%0.130.090.000.070.12 surface

[0066] Table 7. Results of Relative Peak Area (RPA) for the Stability of Test Sample Solutions

[0067] Peak Name Peak 1 Peak 2 Peak 3 (S) Peak 4 50h 3.453 0.522 1.000 0.533 1.7422h 3.433 0.522 1.000 0.534 1.7024h 3.448 0.520 1.000 0.540 1.6856h 3.442 0.518 1.000 0.535 1.7128h 3.43 90.5211.0000.5321.72410h3.4040.5251.0000.5351.69312h3.3250.5301. 0000.5341.69824h3.3690.5221.0000.5331.712RSD%1.330.690.000.461.07 surface

[0068] 5. Sample Determination

[0069] Take appropriate amounts of the finished product (batch numbers 2201, 2202, and 2203), prepare the test solution according to "3. Sample Preparation", and analyze it according to "2. Chromatographic Conditions and System Suitability Conditions". The superimposed characteristic chromatograms of the three batches of Compound Xianghua Granules are shown in Figure 2. With p-coumaric acid as the reference peak S, the relative retention time and relative peak area of ​​each characteristic peak at each detection wavelength are calculated and shown in Tables 8-9.

[0070] Table 8. Results of Relative Retention Time (RRT) of Samples

[0071] Peak Name Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 2201 0.579 0.816 1.000 1.213 1.521 2202 0.577 0.818 1.000 1.217 1.524 2203 0.578 0.818 1.000 1.217 1.522 surface

[0072] Table 9. Results of Relative Retention Peak Area (RPA) for Samples

[0073] Peak number Peak 1 Peak 2 Peak 3 (S) Peak 4 Peak 5 220 13.43 20.52 21.00 0.53 41.73 5 220 23.42 30.51 91.00 0.53 31.72 3 220 33.39 90.52 51.00 0.53 61.71 9 surface

[0074] The HPLC characteristic chromatograms of three batches of Compound Xianghua Granules were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". At a detection wavelength of 310 nm, the reference peak of p-coumaric acid was used as the reference peak, and the reference chromatogram was generated by the median method. The HPLC characteristic fingerprint of Compound Xianghua Granules was established, as shown in Figure 3.

[0075] At a detection wavelength of 310 nm, five characteristic peaks should be detected in the fingerprint chromatogram of Compound Fragrant Flower Granules. Among them, peaks 3 and 4 should correspond to the retention times of the reference peaks of p-coumaric acid and ferulic acid.

[0076] Using the peak corresponding to the reference peak of p-coumaric acid as the S peak, calculate the relative retention time of each characteristic peak and the S peak. The relative retention time of each characteristic peak should be within ±10% of the specified value. The specified values ​​are: 0.58 (peak 1), 0.82 (peak 2), 1.22 (peak 4), and 1.52 (peak 5).

[0077] 6. Conclusion

[0078] The HPLC fingerprint established in this section exhibits uniform retention time distribution of characteristic peaks, good peak symmetry, stable baseline, and satisfactory resolution. Furthermore, the methodology demonstrates good performance, accuracy, and reliability, enabling convenient and rapid overall quality control of finished formulations.

[0079] In summary, this invention, by establishing a fingerprint spectrum for compound fragrant flower granules, can better reflect information on multiple indicative components. The fingerprint spectrum of this invention exhibits stable baseline peaks, good sensitivity and separation, is quick and reliable to operate, and has low cost. It can effectively, stably, and rapidly monitor product quality comprehensively, ensuring product stability and quality controllability.

[0080] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of compound fragrant flower granules, characterized in that, The process includes the following steps: Step 1, preparing the reference solution: dissolving p-coumaric acid and ferulic acid standards in methanol; Step 2, preparing the test solution: dissolving different batches of Compound Xianghua Granules in ultrapure water and performing post-processing; Step 3, injecting the reference solution and the test solution into a high-performance liquid chromatograph (HPLC) for detection, obtaining the fingerprint spectrum of the reference solution and the fingerprint spectrum of each batch of samples; Step 4, analyzing the fingerprint spectrum of each batch of samples using a traditional Chinese medicine fingerprint spectrum similarity evaluation system to generate the finished product fingerprint spectrum of Compound Xianghua Granules; wherein, in Step 3, the HPLC conditions are: column length 100mm-200mm; column inner diameter 3mm-6mm; column packing material octadecylsilane-bonded silica gel with a particle size of 3.0μm-6.0μm; detection wavelength 300nm-320nm; flow rate 0.75-0.85ml / min. n; Detection temperature: 27-33℃; Injection volume: 5-15 μl; Theoretical plate number, calculated based on ferulic acid peak, should be no less than 3000; Elution method: gradient elution; Mobile phase A is acetonitrile, mobile phase B is 0.1% (v / v) phosphoric acid aqueous solution; The gradient elution program includes: 0 min, 5% acetonitrile, 95% (v / v) 0.1% phosphoric acid aqueous solution; 5 min, 5% acetonitrile, 95% (v / v) 0.1% phosphoric acid aqueous solution; 12 min, 12% acetonitrile, 95% (v / v) 0.1% phosphoric acid aqueous solution. Acidic aqueous solution 88%; 20 min, acetonitrile 12%, 0.1% phosphoric acid aqueous solution 88%; 40 min, acetonitrile 30%, 0.1% phosphoric acid aqueous solution 70%; 44 min, acetonitrile 80%, 0.1% phosphoric acid aqueous solution 20%; 46 min, acetonitrile 80%, 0.1% phosphoric acid aqueous solution 20%; 46.1 min, acetonitrile 5%, 0.1% phosphoric acid aqueous solution 95%; 48 min, acetonitrile 5%, 0.1% phosphoric acid aqueous solution 95%.

2. The construction method according to claim 1, characterized in that, In the reference solution, the mass-to-volume ratio of coumaric acid and ferulic acid is 5 μg / mL to 20 μg / mL.

3. The construction method according to claim 1, characterized in that, In the test solution, the ratio of compound fragrant flower granules to ultrapure water is (0.5-2)g:(10-20)mL; the post-processing specifically involves: first ultrasonic treatment, then filtration treatment.

4. The construction method according to claim 3, characterized in that, The ultrasonic treatment has a power of 250W, a frequency of 40kHz, and an ultrasonic time of 30min.

5. The construction method according to claim 1, characterized in that, The fingerprint spectrum of the reference solution includes: peak 3 corresponding to coumaric acid and peak 4 corresponding to ferulic acid.

6. The construction method according to claim 1, characterized in that, The comparative fingerprint spectrum of the compound fragrant flower granules includes 5 characteristic peaks, of which peak 3 is the characteristic peak of p-coumaric acid and peak 4 is the characteristic peak of ferulic acid.

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