A liquid chromatography qualitative and quantitative detection method for centipede formula granules

Abstract

The present invention belongs to the technical field of liquid chromatography analysis of traditional Chinese medicine formula granules, and specifically relates to a liquid chromatography qualitative and quantitative detection method for centipede formula granules. The liquid chromatography qualitative and quantitative detection method for centipede formula granules adopts a methanol aqueous solution with a concentration of 10% to 50% as an extraction solvent, ultrasonic or heated reflux extraction, and the selected reference substances include hypoxanthine, uracil, uridine, and guanosine; the chromatographic column filler is a chromatographic column with octadecylsilane bonded silica gel as a filler, methanol and 0.1% glacial acetic acid solution as a mobile phase, and the detection wavelength is 248nm. The method can present 9 characteristic peaks within 40 minutes of elution time, is suitable for qualitative and quantitative detection, has good adaptability to chromatographic columns, etc., has good specificity, stability, quality control ability, and analysis efficiency, is suitable for one-test-multiple-evaluation quality control, and the method is easy to use and easy to promote.

Description

Technical Field

[0001] The invention belongs to the technical field of liquid chromatography analysis of traditional Chinese medicine formula granules, and particularly relates to a liquid chromatography qualitative and quantitative detection method for centipede formula granules. Background Art

[0002] Centipede is a commonly used animal Chinese medicinal material, pungent, warm and poisonous. It is good at calming wind and relieving spasms, unblocking meridians and relieving pain, and attacking toxins and dispersing knots. It is used for internal liver wind movement, spasms and convulsions, infantile convulsions, stroke, hemiplegia, tetanus, rheumatism, hemiplegia, sores, scrofula, snake and insect bites. The 2020 edition of the Chinese Pharmacopoeia stipulates that the centipede is originally a species of the Scolopendra subspinipes. Scolopendra subspinipesmutilans The dried body of L. Koch.

[0003] Chinese herbal formula granules are granules prepared from single-ingredient Chinese herbal medicine slices using a water extraction process. They retain the characteristics of traditional medication and have the advantages of being easy to carry and easy to prepare, reducing the burden of decocting medicines on patients and medical institutions. Since 2021, with the end of the pilot program in my country, Chinese herbal formula granules have been widely promoted in clinical practice, and their usage has begun to exceed that of Chinese herbal medicine slices. However, some Chinese herbal formula granules, especially those from animal medicinal materials, lack corresponding quality standards and chromatographic identification methods, making their quality control difficult. As far as centipede formula granules are concerned, there are influences such as unclear effective ingredients. At present, my country's pharmacopoeia lacks effective chromatographic analysis methods to conduct quality control such as one test and multiple evaluations of centipede formula granules.

[0004] Domestic scholars and enterprises have conducted exploratory research on this. For example, Xu Yuansheng et al. developed a specific PCR identification method for centipede formula granules (Xu Yuansheng et al. Site-specific PCR identification of centipede formula granules. Chinese Journal of Experimental Traditional Chinese Medicine, 2024, 30(4):48-54.). The gene amplification equipment and technology required for this method have not yet been promoted in pharmaceutical companies and lack a promotion basis. CN202111338155.2 provides an HPLC chromatographic detection method for centipede medicinal materials, targeting hypoxanthine, xanthine and quinoline substances in medicinal materials. The mobile phase is an acetonitrile-ammonium phosphate system, which can detect and analyze 5 substances at the same time, but most of the quinoline substances have not been proven to exist in centipede formula granules. CN202310937609.0 provides an ultra-high performance liquid chromatography (UPLC) detection method for centipede formula granules, the mobile phase is a methanol-potassium dihydrogen phosphate system, which can simultaneously present 6 characteristic peaks, among which 3,8-dihydroxyquinoline is identified, and the characteristic peak retention time exceeds 46 minutes, which is time-consuming, has low detection efficiency, few characteristic peaks, and weak quality control ability. The invention uses a C18 reverse phase column and compares a variety of elution systems, but ultimately abandons the use of a methanol-acetic acid elution system due to factors such as separation, baseline stability, and peak shape, and the methanol-acetic acid elution system has the least chromatographic peaks among the three elution systems. CN202310937552.4 provides a high-performance liquid chromatography (HPLC) detection method for centipede formula granules, the mobile phase is a methanol-water system, which can present 8 characteristic peaks, involving uracil, hypoxanthine, xanthine, inosine, guanosine and some amino acids. And the retention time of the 8 characteristic peaks is within 40 minutes, and the analysis efficiency and quality control ability are improved. However, the chromatographic column adaptability of this method is poor, and only the DikmaPlatisil ODS C18 column meets the requirements.

[0005] So far, there are still few reports on the ingredient analysis and quality control methods of centipede formula granules, which restricts the development of liquid chromatography analysis methods for centipede formula granules. Summary of the invention

[0006] In view of the above problems, the inventors of the present invention unexpectedly found in the performance of research tasks and quality standard research that by using a C18 chromatographic column as the stationary phase, a methanol-glacial acetic acid system as the mobile phase, and a detection wavelength of 248nm, liquid chromatography detection of centipede control medicinal materials and centipede formula granules can present 9 characteristic peaks within a 40-minute elution time, with good peak shape, separation level and baseline stability. This method is suitable for a variety of C18 columns, and has good method specificity, stability and precision. It does not require gene amplification equipment and is suitable for promotion and application in enterprises. The liquid chromatography qualitative detection, quantitative detection method and research and development materials have been reported to the competent authorities for review and filing.

[0007] Therefore, the present invention provides a liquid chromatography qualitative and quantitative detection method for centipede formula granules.

[0008] The centipede refers to the centipede family, which is the giant centipede Scolopendra subspinipes mutilans L. Koch specified in the 2020 edition of the Chinese Pharmacopoeia. The centipede formula granules are the giant centipede Scolopendra subspinipes mutilans L. Koch. Scolopendra subspinipes mutilans The dried body of L. Koch is processed, decocted in water, concentrated, and maltodextrin is added.

[0009] The liquid chromatography qualitative detection method of the centipede formula granules of the present invention comprises the following steps:

[0010] Preparation of test solution: Take centipede formula granules, use 10%~50% methanol aqueous solution for ultrasonic or heating reflux extraction, filter, and take the filtrate as the test solution;

[0011] Preparation of reference solution: Take hypoxanthine reference, uracil reference, uridine reference and guanosine reference, and dissolve them in methanol-water solution to prepare reference solution;

[0012] Injection test: Take the test solution and the reference solution for injection respectively; the chromatographic column is filled with octadecylsilane bonded silica gel, and the chromatographic column parameters are: column length 250mm, inner diameter 4.6mm, particle size 5μm; methanol is used as mobile phase A, 0.1% glacial acetic acid solution is used as mobile phase B, and gradient elution is performed; the flow rate is 0.9mL~1.1mL per minute, the column temperature is 28℃~32℃, and the detection wavelength is 248nm; the elution program includes the following procedures: 0~15 minutes: the volume percentage of mobile phase A is 0%; 15 minutes~30 minutes: the volume percentage of mobile phase A increases from 0% to 10%; 30~45 minutes: the volume percentage of mobile phase A increases from 10% to 40%.

[0013] Preferably, in the test solution preparation step, the extraction time is 15 minutes to 45 minutes.

[0014] Preferably, in the step of preparing the test solution, the extraction method is ultrasonic extraction; the ultrasonic power is 400W and the frequency is 40kHz.

[0015] Preferably, in the step of preparing the reference solution, the concentration of the methanol aqueous solution is 10% to 30%.

[0016] Preferably, in the reference solution preparation step, the concentrations of hypoxanthine, uracil, uridine and guanosine in the reference solution are 10 μg, 5 μg, 8 μg and 8 μg per mL, respectively.

[0017] Preferably, in the test solution preparation step, the extraction solvent is a 30% methanol aqueous solution.

[0018] Preferably, the qualitative detection method further comprises the following steps:

[0019] Qualitative identification of centipede formula granules: Analyze the chromatogram obtained in the sampling and detection steps. There should be 9 characteristic peaks in the chromatogram of the test sample. Peak 1, peak 4, peak 6, and peak 8 correspond to the retention time of uracil, hypoxanthine, uridine, and guanosine chromatographic peaks. The peak corresponding to the hypoxanthine reference peak, peak 4, is taken as the S1 peak. The relative retention time of peaks 2, 3, and 5 with the S1 peak is calculated. The relative retention time should be within ±10% of the specified value. The specified value is: 0.66 (peak 2), 0.90 (peak 3), and 1.24 (peak 5). The peak corresponding to the guanosine reference peak, peak 8, is taken as the S2 peak. The relative retention time of peaks 7 and 9 with the S2 peak is calculated. The relative retention time should be within ±10% of the specified value. The specified value is: 0.97 (peak 7) and 1.29 (peak 9).

[0020] The liquid chromatography quantitative detection method of centipede formula granules of the present invention comprises the following steps:

[0021] Preparation of test solution: Take centipede formula granules, use 10%~50% methanol aqueous solution for ultrasonic or heating reflux extraction, filter, and take the filtrate as the test solution;

[0022] Preparation of reference solution: Dissolve hypoxanthine reference substance in methanol-water solution to prepare hypoxanthine reference solution;

[0023] Injection test: Take the test solution and the hypoxanthine reference solution and inject them respectively to determine the hypoxanthine content in the test solution; the chromatographic column is filled with octadecylsilane bonded silica gel, and the chromatographic column parameters are: column length 250mm, inner diameter 4.6mm, particle size 5μm; methanol is used as mobile phase A, 0.1% glacial acetic acid solution is used as mobile phase B, and gradient elution is performed; the flow rate is 1.0mL per minute, the column temperature is 30℃, and the detection wavelength is 248nm; the elution program includes the following procedures: 0~15 minutes: the volume percentage of mobile phase A is 0%; 15 minutes~30 minutes: the volume percentage of mobile phase A increases from 0% to 10%; 30~45 minutes: the volume percentage of mobile phase A increases from 10% to 40%.

[0024] Preferably, in the test solution preparation step, the extraction time is 15 minutes to 45 minutes.

[0025] Preferably, in the step of preparing the test solution, the extraction method is ultrasonic extraction; the ultrasonic power is 400W and the frequency is 40kHz.

[0026] Preferably, in the step of preparing the reference solution, the concentration of the methanol aqueous solution is 10% to 30%.

[0027] Preferably, the concentration of hypoxanthine in the hypoxanthine reference solution is 10 μg per mL.

[0028] Preferably, in the test solution preparation step, the extraction solvent is a 30% methanol aqueous solution.

[0029] The solubility and solubility of uracil, hypoxanthine, uridine and guanosine are common knowledge in the art. Those skilled in the art can select different methanol-water solutions according to the solubility and solubility of the reference substance, and adjust the concentration of the reference substance in the solution. For example, CN202310937552.4 uses 30% methanol as the solvent, and the concentration of the reference substance in the solution is 0.1 mg / mL. Beneficial Effects

[0030] (1) The chromatographic columns of this liquid chromatography detection method have good adaptability: JADE-PAK ODS-AQ (4.6 mm × 250 mm, 5 μm) and COSMOSIL Packed Column 5C18-MS-Ⅱ (4.6 mm × 250 mm, 5 μm) both types of C18 columns meet the analysis requirements;

[0031] (2) The instrumentation and equipment for this liquid chromatography detection method are well adaptable: both Waters 2695 and UltiMate 3000 high performance liquid chromatographs meet the analysis requirements;

[0032] (3) The extraction conditions of this liquid chromatography detection method are well adaptable: the sample is extracted with a methanol aqueous solution with a concentration of 10% to 50% by ultrasonic or heating reflux for 15 minutes to 45 minutes to obtain a chromatogram that meets the analysis requirements;

[0033] (4) The quality control capability and analytical efficiency of this liquid chromatography detection method are good: 9 characteristic peaks can be obtained within a 40-minute elution time, which makes this method have high quality control capability and high analytical efficiency;

[0034] (5) The characteristic peaks of this liquid chromatography detection method have good separation, good peak shape, and a stable baseline. It has both qualitative and quantitative identification capabilities and is suitable for multiple evaluations with one test. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a comparison of the characteristic spectra of the two extraction methods of the centipede formula granules in Example 2;

[0036] Figure 2 This is a comparison of characteristic spectra of different extraction solvents of centipede formula granules in Example 2;

[0037] Figure 3 This is a comparison diagram of the characteristic spectra of the centipede formula granules at different extraction times in Example 2;

[0038] Figure 4 It is the characteristic spectrum specificity investigation spectrum of the centipede formula granules in Example 3;

[0039] Figure 5 The characteristic spectrum of the centipede formula granules in Example 3 is a double running time spectrum;

[0040] Figure 6 This is a stacked diagram of the characteristic spectrum of the centipede formula granules in Example 3 for sampling precision inspection;

[0041] Figure 7 This is a stacked diagram of the repeatability of the characteristic spectrum of the centipede formula granules in Example 3;

[0042] Figure 8 It is a stacked diagram of the intermediate precision inspection of the characteristic diagram of the centipede formula granules in Example 3;

[0043] Fig. 9 This is a stacked diagram of the stability study of the characteristic diagram of the centipede formula granules in Example 3;

[0044] Fig.10 This is a stacked diagram of the characteristic diagram of the centipede formula granules in Example 3 at different column temperatures;

[0045] Fig.11 This is a stacked diagram of the characteristic diagram of the centipede formula granules in Example 3 at different flow rates;

[0046] Fig.12 The characteristic diagram of centipede formula granules in Example 3 is not stacked with chromatographic columns Figure 1 ;

[0047] Fig.13 The characteristic diagram of centipede formula granules in Example 3 is not stacked with chromatographic columns Figure 2 ;

[0048] Fig.14 It is a stacked diagram of the characteristic spectra of three batches of centipede formula granules in Example 4;

[0049] Fig.15 The reference characteristic spectrum of the centipede formula granules established in Example 4;

[0050] Fig.16 It is a graph showing the specificity of the content determination of the centipede formula granules in Example 5;

[0051] Fig.17 The spectra of twice the running time were investigated for the specificity of the content determination of the centipede formula granules in Example 5;

[0052] Fig.18This is the peak purity inspection spectrum for the content determination of the centipede formula granules in Example 5;

[0053] In the figure, peak 1 is uracil, peak 4 is hypoxanthine, peak 6 is uridine, and peak 8 is guanosine. DETAILED DESCRIPTION

[0054] The following is an explanation of the centipede formula granule identification method of the present invention through specific examples. The embodiments are illustrative examples and are not to be construed as limiting the scope of protection. Unless otherwise specified, the methanol concentration in the embodiments is a volume ratio, i.e., a volume percentage concentration. For example, 10%, 30%, and 50% methanol as an extraction solvent are 10%, 30%, and 50% methanol aqueous solutions, respectively. Unless otherwise specified, the glacial acetic acid concentration in the embodiments is a volume percentage concentration. For example, a 0.1% glacial acetic acid solution as mobile phase B is a 0.1% volume percentage glacial acetic acid aqueous solution. Unless otherwise specified, in the embodiments, RAD is the relative average deviation, RSD is the relative standard deviation, and SD is the standard deviation. Information on instruments, reagents, etc. used in the embodiments of the present invention is as follows:

[0055] Chromatographic columns: Column 1: JADE-PAK ODS-AQ 4.6mm×250mm, 5μm (Batch number: US2237A2UE93); Column 2: JADE-PAK ODS-AQ 4.6mm×250mm, 5μm (Batch number: US2137AJUD73); Column 3: COSMOSILPacked Column 5C18-MS-Ⅱ 4.6mm×250mm, 5μm (Batch number: Manf No K66594).

[0056] Reagents: Methanol was of chromatographic grade and produced by Chengdu Kelong Chemicals Co., Ltd.; glacial acetic acid was of chromatographic grade and produced by Chengdu Kelong Chemicals Co., Ltd.; water was purified water prepared by an ultrapure water machine.

[0057] Test drugs: hypoxanthine reference substance (batch number: 140661-202005, purity: 99.4%, China Food and Drug Inspection Institute); uracil reference substance (batch number: PS020117, purity: 98.0%, Chengdu Pusi Biotechnology Co., Ltd.); uridine (batch number: PS010290, purity: 98.0%, Chengdu Pusi Biotechnology Co., Ltd.); guanosine (batch number: PS010291, purity: 98.0%, Chengdu Pusi Biotechnology Co., Ltd. Co., Ltd.); centipede control medicinal material (batch number: DST210911-013, Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.); centipede formula granules, prepared by the applicant, preparation method: take 3000g of centipede slices, add water to boil, filter, concentrate the filtrate into a clear paste (the dry extract yield is 17.0% to 31.0%), add auxiliary material maltodextrin, dry (or dry and crush), mix, granulate, and make 1000g, that is, 1g of centipede formula granules is equivalent to 3g of slices.

[0058] Instruments and equipment: High performance liquid chromatograph 1 (Waters2695, Waters Technology (Shanghai) Co., Ltd.); High performance liquid chromatograph 2 (UltiMate3000, Thermo Fisher Scientific (China) Co., Ltd.); 1 / 100 analytical balance (CN-LQC10002, Kunshan Youkuvit Electronic Technology Co., Ltd.); 1 / 10,000 analytical balance (FA1004, Lichen Technology Co., Ltd.); 1 / 100,000 analytical balance (AUW220D, Shimadzu Corporation, Japan); CNC ultrasonic cleaner (KH-500DE, Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); centrifuge (TG-16B, Changzhou Zhengrong Instrument Co., Ltd.); ultrapure water system (EKUP-II-10p, Sichuan Yike Pure Water Equipment Co., Ltd.); electronic temperature control electric heating jacket (DZTW, Hangzhou Jingfei Instrument Technology Co., Ltd.).

[0059] The purpose of preparing the hypoxanthine reference solution separately in the following examples is to facilitate the reference between the methods of Example 2 and Example 5 and to facilitate the separate comparison of methodological investigations. When the liquid chromatography qualitative detection of the centipede formula granules is performed, hypoxanthine, uracil, uridine, and guanosine can be mixed in the same solution as the reference solution.

[0060] Example 1 Preparation of centipede formula granules

[0061] The preparation of centipede formula granules comprises the following steps:

[0062] Preparation: Take centipede medicinal material, remove the bamboo strips, wash, roast on low heat until yellow, and cut into sections.

[0063] Extraction: Weigh centipede slices, add water and boil twice. For the first time, add 8 times the amount of water (w / w), boil for 1 hour and filter; for the second time, add 6 times the amount of water (w / w), boil for 0.5 hour, filter and combine the filtrate.

[0064] Concentration: Take the filtrate and concentrate it under reduced pressure at 80℃ to a clear paste with a relative density of 1.01-1.06 (80℃).

[0065] Vacuum drying: take the clear paste, add maltodextrin, vacuum dry (water tank temperature 80-90℃, box temperature 40-60℃), crush, collect powder, sieve, and get dry paste powder.

[0066] Total mixing: Put the dry paste powder and the calculated amount of sieved maltodextrin into a mixer and mix them evenly to obtain the total mixed powder. Calculated amount of maltodextrin = amount of decoction pieces / 3-amount of dry paste powder

[0067] Dry granulation: take the total mixed powder, dry granulate, granulate, and obtain granules. 1g of this formula granules is equivalent to 3g of decoction pieces.

[0068] Example 2 Investigation of extraction conditions for qualitative detection

[0069] Instruments and equipment: High performance liquid chromatograph 1.

[0070] 1. Chromatographic conditions

[0071] Octadecylsilane bonded silica gel was used as the filler (chromatographic column 1); methanol was used as the mobile phase A, 0.1% glacial acetic acid solution was used as the mobile phase B, and elution was performed according to the procedure in Table 1; the flow rate was 1.0 mL per minute; the column temperature was 30°C; the detection wavelength was 248 nm, and the injection volume was 10 μL. The theoretical plate number was calculated based on the hypoxanthine peak and was not less than 10,000.

[0072] Table 1 Gradient elution program

[0073]

[0074] 2. Preparation of reference solution

[0075] 2.1 Preparation of reference medicinal material solution: Take 0.2 g of centipede reference medicinal material, place it in a stoppered conical flask, add 25 mL of 30% methanol, weigh it, heat and reflux for 15 minutes, cool it, weigh it again, make up the lost weight with 30% methanol, shake it well, take the supernatant, filter it, and take the filtrate as the reference medicinal material solution.

[0076] 2.2 Preparation of hypoxanthine reference solution: Take hypoxanthine reference, weigh it, add 10% methanol to make a solution containing 10 μg per mL.

[0077] 2.3 Preparation of mixed solution of uracil, uridine and guanosine reference substances: Weigh uracil reference substance, uridine reference substance and guanosine reference substance, add 10% methanol to prepare a mixed solution containing 5 μg uracil, 8 μg uridine and 8 μg guanosine per mL as the mixed solution of reference substances.

[0078] 3. Determination and chromatographic qualitative identification

[0079] Respectively take 10 μL of reference solution (i.e., reference medicinal material reference solution, hypoxanthine reference solution, and reference reference mixed solution) and test solution, inject into liquid chromatograph, and measure to obtain the result.

[0080] Chromatographic qualitative identification: According to this method, the chromatogram of the test sample should show 9 characteristic peaks, which correspond to the retention times of the 9 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 1, peak 4, peak 6, and peak 8 correspond to the retention times of uracil, hypoxanthine, uridine, and guanosine chromatographic peaks, respectively. The peak corresponding to the hypoxanthine reference peak, peak 4, is taken as the S1 peak, and the relative retention time of peak 2, peak 3, peak 5 and S1 peak is calculated. The relative retention time should be within ±10% of the specified value, and the relative retention time specified value is: 0.66 (peak 2), 0.90 (peak 3), 1.24 (peak 5). The peak corresponding to the guanosine reference peak, peak 8, is taken as the S2 peak, and the relative retention time of peak 7, peak 9 and S2 peak is calculated. The relative retention time should be within ±10% of the specified value, and the relative retention time specified value is: 0.97 (peak 7), 1.29 (peak 9).

[0081] 4. Investigation of the pretreatment method of the test sample

[0082] 4.1 Investigation of extraction methods

[0083] Take centipede formula granules, grind, take about 0.2g (two groups in total), weigh, put in a stoppered conical flask, add 25mL of 10% methanol, stopper, weigh, ultrasonically treat (power 400W, frequency 40kHz) or heat reflux for 30 minutes, cool, weigh again, make up the lost weight with 10% methanol, shake well, filter, and take the filtrate. Analyze the peak shape and separation of the 9 characteristic peaks, and calculate the "total peak area / sample amount" of the 9 characteristic peaks to compare the effects of different extraction methods on the chromatogram. The results are shown in Table 2 and Figure 1 The results showed that there was no significant difference in the chromatographic peaks and "total peak area / sample weight" between ultrasonic and reflux extraction. Ultrasonic extraction was more convenient, so ultrasonic treatment was selected as the extraction method.

[0084] Table 2 Characteristic spectrum of centipede formula granules investigated by different extraction methods

[0085]

[0086] 4.2 Investigation of extraction solvent

[0087] Preliminary experiments have shown that methanol extraction is better than ethanol. This experiment examined the extraction effects of different concentrations of methanol.

[0088] Take centipede formula granules, grind, take about 0.2g (three groups in total), weigh, put in a stoppered conical bottle, add 25mL of 10% methanol, 30% methanol, and 50% methanol respectively, weigh, ultrasonically treat (power 400W, frequency 40kHz) for 30 minutes, cool, weigh again, add corresponding solvents to make up for the loss, shake well, filter, and take the filtrate. Analyze the peak shape and separation of the 9 characteristic peaks, and calculate the "total peak area / sample amount" of the 9 characteristic peaks to compare the effects of different extraction solvents on the chromatogram. The results are shown in Table 3 and Figure 2 The results showed that there was no significant difference in the chromatographic peaks and "total peak area / sample weight" extracted with different solvents. Combined with the method of Example 5, 30% methanol was selected as the extraction solvent.

[0089] Table 3 Characteristic spectrum of centipede formula granules extraction solvent investigation

[0090]

[0091] 4.3 Extraction time investigation

[0092] Take centipede formula granules, grind, take about 0.2g (three groups in total), weigh, put in a stoppered conical flask, add 25mL of 30% methanol, stopper, weigh, ultrasonically treat (power 400W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, cool, make up the lost weight with 30% methanol, shake well, filter, and take the filtrate. Analyze the peak shape and separation of the 9 characteristic peaks, and calculate the "total peak area / sample amount" of the 9 characteristic peaks to compare the effects of different extraction times on the chromatogram. The results are shown in Table 4. Figure 3 The results showed that there was no significant difference in the chromatographic peaks and "total peak area / sample weight" extracted at different extraction times. Therefore, 15 minutes was selected as the extraction time.

[0093] Table 4 Characteristic spectrum of centipede formula granules at different extraction times

[0094]

[0095] 4.4 Determination of the preparation method of the test solution

[0096] like Figures 1 to 3 The retention times (minutes) of peaks 1 to 9 are 6.81~6.85, 7.64~8.01, 10.59~10.64, 11.75~11.80, 14.56~14.60, 16.54~16.88, 28.83~28.99, 29.61~29.76, and 38.65~39.23, respectively.

[0097] In summary, the preparation method of the test solution is determined as follows: take the centipede formula granules, grind, take about 0.2g, weigh, put it in a stoppered conical flask, add 25mL of 30% methanol, stopper, weigh the weight, ultrasonically treat (power 400W, frequency 40kHz) for 15 minutes, cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, and take the filtrate to obtain the solution.

[0098] Example 3 Methodological verification of qualitative detection

[0099] 1. Exclusive inspection

[0100] The auxiliary material of centipede formula granules is maltodextrin. This experiment investigates the effect of maltodextrin on qualitative detection. Take maltodextrin and prepare a negative sample solution according to the test solution preparation method under "4.4" of Example 2. Take the test solution, negative sample solution, and reference solution, and analyze them according to the instrument of Example 2 and the chromatographic conditions under "1" of Example 2, and record the chromatogram. At the same time, continue to run the same elution time according to the endpoint gradient ratio, examine the chromatographic peak after the specified time, and record the chromatogram. The results are shown in Figure 4 , Figure 5 The results showed that the test sample presented 9 characteristic peaks, which corresponded to the retention time of the 9 characteristic peaks of the control medicinal material. Among them, peaks 1, 4, 6, and 8 corresponded to the retention time of the chromatographic peaks of uracil, hypoxanthine, uridine, and guanosine. Maltodextrin had no interference with the chromatogram, and there were no other chromatographic peaks in the test sample after 60 minutes. This method is specific.

[0101] 2. Precision inspection

[0102] 2.1 Investigation of injection precision

[0103] Take centipede formula granules, grind, take about 0.2g, weigh, prepare test solution according to Example 2 "4.4", sample analysis according to the instrument of Example 2 and the chromatographic conditions under Example 2 "1", inject the same sample solution 6 times continuously, the peak corresponding to the hypoxanthine reference peak, that is, peak 4, is the S1 peak, calculate the relative retention time of peak 2, peak 3, peak 5 and S1 peak; the peak corresponding to the guanosine reference peak, that is, peak 8, is the S2 peak, calculate the relative retention time of peak 7, peak 9 and S2 peak; calculate the relative peak area of ​​peak 1 to peak 3, peak 5 to peak 9 and S1 peak, and calculate the RSD value. The results are shown in Table 5, Table 6, Figure 6 The results showed that the relative retention time RSD range of peak 2, peak 3, peak 5 and peak S1 was 0.04%~0.12%; the relative retention time RSD values ​​of peak 7, peak 9 and peak S2 were 0.00%~0.09%; the relative peak area RSD values ​​of peak 1~peak 3, peak 5~peak 9 and peak S1 were 0.38%~2.25%; the RSD values ​​were all <5%, and the instrument precision was good.

[0104] Table 5 Results of precision study of characteristic spectrum of centipede formula granules (relative retention time)

[0105]

[0106] Table 6 Results of precision study on characteristic spectrum of centipede formula granules (relative peak area)

[0107]

[0108] 2.2 Repeatability study

[0109] Take centipede formula granules, grind, take about 0.2g, weigh 6 parts in parallel, prepare the test solution according to Example 2 "4.4", and analyze the sample according to the instrument of Example 2 and the chromatographic conditions under Example 2 "1". The peak corresponding to the hypoxanthine reference peak, that is, peak 4, is the S1 peak, and the relative retention time of peak 2, peak 3, peak 5 and S1 peak is calculated; the peak corresponding to the guanosine reference peak, that is, peak 8, is the S2 peak, and the relative retention time of peak 7, peak 9 and S2 peak is calculated; the relative peak area of ​​peak 1 to peak 3, peak 5 to peak 9 and S1 peak is calculated, and the RSD value is calculated. The results are shown in Table 7, Table 8, Figure 7 The results showed that the relative retention time RSD range of peak 2, peak 3, peak 5 and peak S1 was 0.03%~0.64%; the relative retention time RSD values ​​of peak 7, peak 9 and peak S2 were 0.00%~0.17%; the relative retention time RSD values ​​were all <5%, and the relative peak area RSD values ​​of peak 1~peak 3, peak 5~peak 9 and peak S1 were 0.31%~2.67%; the relative peak area RSD values ​​were all <5%, and the qualitative method had good repeatability.

[0110] Table 7 Results of repeatability study of characteristic spectra of centipede formula granules (relative retention time)

[0111]

[0112] Table 8 Results of repeatability study of characteristic spectrum of centipede formula granules (relative peak area)

[0113]

[0114] 2.3 Intermediate precision study

[0115] Different analysts operated on different instruments at different times, took centipede formula granules, ground them, took about 0.2g, weighed 6 portions in parallel, prepared the test solution according to "4.4" of Example 2, injected and analyzed according to the chromatographic conditions under "1" of Example 2, the peak corresponding to the hypoxanthine reference peak, that is, peak 4, was the S1 peak, and the relative retention time of peaks 2, 3, and 5 with the S1 peak was calculated; the peak corresponding to the guanosine reference peak, that is, peak 8, was the S2 peak, and the relative retention time of peaks 7 and 9 with the S2 peak was calculated; the relative peak areas of peaks 1 to 3, peaks 5 to 9 with the S1 peak were calculated, and the RSD value was calculated. The results are shown in Tables 9, 10, Figure 8 . The experimental results show that personnel B and personnel A operated on different instruments at different times. The relative retention time RSD range of peak 2, peak 3, peak 5 and S1 peak was 0.03%~1.52%; the relative retention time RSD values ​​of peak 7, peak 9 and S2 peak were 0.08%~0.64%; the relative retention time RSD values ​​were all <5%. The relative peak area RSD values ​​of peak 1~peak 3, peak 5~peak 9 and S1 peak were 1.87%~7.49%; the relative peak area RSD values ​​of peak 1 and peak 3 were all >5%. The intermediate precision of relative retention time of this qualitative method is good.

[0116] Table 9 Intermediate precision test results of centipede formula granules (relative retention time)

[0117]

[0118] Table 10 Intermediate precision investigation results of centipede formula granules (relative peak area)

[0119]

[0120] In Tables 9 and 10, repeatability: Personnel A, instrument: high performance liquid chromatograph 1, number: JS-01-007; experimental date: 2022 / 05 / 06; intermediate precision: Personnel B, instrument: high performance liquid chromatograph 2, number: JS-01-012; experimental date: 2022 / 05 / 09.

[0121] 3. Stability test

[0122] Take centipede formula granules, grind, take about 0.2g, weigh, prepare test solution according to Example 2 "4.4", sample analysis according to the instrument of Example 2 and the chromatographic conditions under Example 2 "1", sample analysis at 0, 10, 30, 40, 52 hours respectively, the peak corresponding to the hypoxanthine reference peak, that is, peak 4, is the S1 peak, calculate the relative retention time of peak 2, peak 3, peak 5 and S1 peak; the peak corresponding to the guanosine reference peak, that is, peak 8, is the S2 peak, calculate the relative retention time of peak 7, peak 9 and S2 peak; calculate the relative peak area of ​​peak 1 to peak 3, peak 5 to peak 9 and S1 peak, and calculate the RSD value. The results are shown in Table 11, Table 12, Fig. 9 The results showed that the relative retention time RSD range of peak 2, peak 3, peak 5 and peak S1 was 0.09%~0.26%; the relative retention time RSD values ​​of peak 7, peak 9 and peak S2 were 0.00%~0.33%; the relative peak area RSD values ​​of peak 1~peak 3, peak 5~peak 9 and peak S1 were 0.54%~3.18%; the RSD of relative retention time and relative peak area were both <5%, indicating that the test solution was stable and measurable after being placed for 52 hours.

[0123] Table 11 Results of stability study of characteristic spectrum of centipede formula granules (relative retention time)

[0124]

[0125] Table 12 Results of stability study of characteristic spectrum of centipede formula granules (relative peak area)

[0126]

[0127] 4. Durability inspection

[0128] 4.1 Investigation of different column temperatures

[0129] Compare the effects of different column temperatures, 28°C, 30°C and 32°C on the chromatogram. Take the centipede formula granules, grind, take about 0.2g, weigh, and prepare the test solution according to Example 2 "4.4". Except for the column temperature, the other chromatographic conditions are the same as the instrument of Example 2 and the chromatographic conditions of Example 2 "1". Sample injection analysis, the peak corresponding to the hypoxanthine reference peak, that is, peak 4, is the S1 peak, and the relative retention time of peaks 2, 3, and 5 with the S1 peak is calculated; the peak corresponding to the guanosine reference peak, that is, peak 8, is the S2 peak, and the relative retention time of peaks 7 and 9 with the S2 peak is calculated; the relative peak areas of peaks 1 to 3, peaks 5 to 9 and the S1 peak are calculated, and the RSD value is calculated. The results are shown in Table 13, Table 11, Fig.10The results showed that the relative retention time RSD range of peak 2, peak 3, peak 5 and peak S1 was 0.33%~0.80%; the relative retention time RSD values ​​of peak 7, peak 9 and peak S2 were 0.00%~1.82%; the relative peak area RSD values ​​of peak 1~peak 3, peak 5~peak 9 and peak S1 were 1.14%~5.34%; the relative retention time RSD was <5%, and the relative peak area RSD of peak 8 was >5%, indicating that when the column temperature varied slightly within the range of ±2℃, the relative retention time durability of the qualitative method was good.

[0130] Table 13 Column temperature investigation results of centipede formula granule characteristic spectrum (relative retention time)

[0131]

[0132] Table 14 Column temperature investigation results of centipede formula granule characteristic spectrum (relative peak area)

[0133]

[0134] 4.2 Investigation of different flow rates

[0135] Compare the effects of different flow rates (0.9mL / min, 1.0mL / min and 1.1mL / min) on the chromatogram. Take the centipede formula granules, grind, take about 0.2g, weigh, and prepare the test solution according to Example 2 "4.4". Except for the flow rate, the other chromatographic conditions are the same as the instrument of Example 2 and the chromatographic conditions of Example 2 "1". Sample injection analysis, the peak corresponding to the hypoxanthine reference peak, that is, peak 4, is the S1 peak, and the relative retention time of peaks 2, 3, and 5 with the S1 peak is calculated; the peak corresponding to the guanosine reference peak, that is, peak 8, is the S2 peak, and the relative retention time of peaks 7 and 9 with the S2 peak is calculated; calculate the relative peak areas of peaks 1 to 3, peaks 5 to 9 and the S1 peak, and calculate the RSD value. The results are shown in Tables 15, 16, Fig.11 The results showed that the relative retention time RSD range of peak 2, peak 3, peak 5 and peak S1 was 0.23%~0.64%; the relative retention time RSD values ​​of peak 7, peak 9 and peak S2 were 0.10%~0.62%; the relative peak area RSD values ​​of peak 1~peak 3, peak 5~peak 9 and peak S1 were 0.84%~3.35%; the relative retention time and relative peak area RSD values ​​were <5%, indicating that when the flow rate changed slightly within the range of ±0.1mL / min, the qualitative method had good durability for relative retention time and relative peak area.

[0136] Table 15 Flow rate investigation results of centipede formula granule characteristic spectrum (relative retention time)

[0137]

[0138] Table 16 Flow rate investigation results of centipede formula granule characteristic spectrum (relative peak area)

[0139]

[0140] 4.3 Investigation of different chromatographic columns

[0141] Compare the effects of chromatographic columns 1, 2, and 3 on the chromatogram. Take the centipede formula granules, grind them, take about 0.2g, weigh them, and prepare the test solution according to "4.4" of Example 2. Refer to the instrument of Example 2 and the chromatographic conditions under "1" of Example 2 for determination. The peak corresponding to the hypoxanthine reference peak, i.e., peak 4, is the S1 peak. Calculate the relative retention time of peaks 2, 3, 5, and S1; the peak corresponding to the guanosine reference peak, i.e., peak 8, is the S2 peak. Calculate the relative retention time of peaks 7 and 9 and S2; Calculate the relative peak areas of peaks 1 to 3, peaks 5 to 9, and S1, and calculate the RAD value. The comparison results of chromatographic columns 1 and 2 are shown in Tables 17, 18, and Fig.12 ; The comparison results of chromatograms 1 and 3 are shown in Table 19, Table 20, Fig.13 .

[0142] Table 17 Characteristic spectrum of centipede formula granules chromatographic column investigation results 1 (relative retention time)

[0143]

[0144] Table 18 Characteristic spectrum of centipede formula granules chromatographic column investigation results 1 (relative peak area)

[0145]

[0146] From Table 17, Table 18, Fig.12 It can be seen that the relative retention time RAD range of peak 2, peak 3, peak 5 and peak S1 is 0.06%~0.68% for the same model but different batches of chromatographic columns; the relative retention time RAD value of peak 7, peak 9 and peak S2 is 0.05%~0.31%; the relative peak area RAD value of peak 1~peak 3, peak 5~peak 9 and peak S1 is 0.27%~8.11%; the relative retention time RAD is <5%. This qualitative method has good durability for different batches of JADE-PAK® ODS-AQ chromatographic columns.

[0147] Table 19 Characteristic spectrum of centipede formula granules chromatographic column investigation results 2 (relative retention time)

[0148]

[0149] Table 20 Characteristic spectrum of centipede formula granules chromatographic column investigation results 2 (relative peak area)

[0150]

[0151] From Table 19, Table 20, Fig.13 It can be seen that the relative retention time RAD range of peak 2, peak 3, peak 5 and S1 peak was 0.12%~0.82% when measured by two different types of chromatographic columns; the relative retention time RAD value of peak 7, peak 9 and S2 peak was 0.21%~0.80%; the relative peak area RAD value of peak 1~peak 3, peak 5~peak 9 and S1 peak was 1.82%~18.37%; the relative retention time RAD was <5%, and the qualitative method had good durability for the relative retention time of different types of chromatographic columns.

[0152] Example 4 Qualitative testing of samples and formulation of qualitative identification standards

[0153] Take 3 batches of centipede formula granules (572303001a, 572303002a, 572303003a), and prepare the test solution according to Example 2 "4.4". Take the test solution and the centipede control reference solution respectively, and measure according to the instrument of Example 2 and the chromatographic conditions under Example 2 "1". The peak corresponding to the hypoxanthine reference peak, that is, peak 4, is the S1 peak, and the relative retention time of peak 2, peak 3, peak 5 and S1 peak is calculated; the peak corresponding to the guanosine reference peak, that is, peak 8, is the S2 peak, and the relative retention time of peak 7, peak 9 and S2 peak is calculated; the relative peak areas of peaks 1 to 3, peaks 5 to 9 and S1 peak are calculated, and the RSD value is calculated. The measurement results are shown in Table 21, Table 22, Fig.14 The HPLC characteristic spectra of three batches of large-scale production of centipede formula granules were matched using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and a reference spectrum was generated by the average method to establish a reference characteristic spectrum of centipede formula granules, see Fig.15 The results showed that the characteristic spectra of the three batches of large-scale production of centipede formula granules had the same 9 characteristic peaks, and the characteristic spectrum standard of centipede formula granules (i.e., the qualitative identification standard of liquid chromatography qualitative detection) was determined as follows:

[0154] There should be 9 characteristic peaks in the chromatogram of the test product. Peak 1, peak 4, peak 6, and peak 8 correspond to the retention times of uracil, hypoxanthine, uridine, and guanosine chromatographic peaks. The peak corresponding to the hypoxanthine reference peak, peak 4, is the S1 peak. The relative retention times of peaks 2, 3, and 5 with the S1 peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.66 (peak 2), 0.90 (peak 3), and 1.24 (peak 5). The peak corresponding to the guanosine reference peak, peak 8, is the S2 peak. The relative retention times of peaks 7 and 9 with the S2 peak are calculated. The relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.97 (peak 7) and 1.29 (peak 9).

[0155] Table 21 Characteristic spectra of centipede formula granules (relative retention time)

[0156]

[0157] Table 2 Characteristic spectra of 2 batches of centipede formula granules (relative peak area)

[0158]

[0159] comprehensive Figures 1 to 15 The chromatogram and the corresponding relative retention time data analysis show that this qualitative method has high analysis efficiency (9 characteristic peaks are obtained within 40 minutes of elution time) and high quality control capability (9 characteristic peaks can be analyzed and identified simultaneously); and the characteristic peaks have good separation to meet the needs of qualitative analysis, good peak shape, and a stable baseline.

[0160] Example 5 Sample Quantitative Determination and Methodological Study of Quantitative Determination

[0161] The sample quantitative detection object is set as hypoxanthine, and the chromatographic conditions for quantitative determination, preparation of the test sample, and preparation of the hypoxanthine reference solution are the same as in Example 2.

[0162] 1. Investigation of the pretreatment method of the test sample

[0163] 1.1 Investigation of extraction methods

[0164] Take the centipede formula granules, grind, take about 0.2g (two groups in total), weigh, put in a stopper conical flask, add 25mL of 10% methanol, plug, weigh, respectively investigate ultrasonic treatment (power 400W, frequency 40kHz), heating reflux two methods, extraction time is 30 minutes, cool, weigh again, make up the weight lost with 10% methanol, shake well, filter, take the subsequent filtrate as the test solution. Determine the hypoxanthine content according to the instrument of Example 2 and the chromatographic conditions under the item "1" of Example 2. As shown in Table 23, the content of hypoxanthine obtained by the two extraction methods is basically the same. Because ultrasonic extraction is convenient to operate, ultrasonic extraction is selected as the extraction method for the determination of the centipede formula granules.

[0165] Table 23 Investigation of different extraction methods for content determination of centipede formula granules

[0166]

[0167] 1.2 Investigation of extraction solvent

[0168] Take the centipede formula granules, grind, take about 0.2g (three groups in total), weigh, put in a stoppered conical flask, add 25mL of 10% methanol, 30% methanol, and 50% methanol respectively, weigh, ultrasonically treat (power 400W, frequency 40kHz) for 30 minutes, cool, weigh again, add corresponding solvents to make up for the weight loss, shake well, filter, and take the filtrate as the test solution. Determine the hypoxanthine content according to the instrument of Example 2 and the chromatographic conditions under item "1" of Example 2. As shown in Table 24, there is no obvious difference in the hypoxanthine content extracted by three concentrations of methanol. Comprehensive test results and the convenience of experimental operation, select 30% methanol as the extraction solvent for the centipede granule content determination.

[0169] Table 24 Centipede formula granule content determination extraction solvent investigation

[0170]

[0171] 1.3 Extraction time investigation

[0172] Take the centipede formula granules, grind, take about 0.2g (three groups in total), weigh, put in a stoppered conical flask, add 25mL of 30% methanol, plug, weigh, ultrasonically treat (power 400W, frequency 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, cool, make up the lost weight with 30% methanol, shake well, filter, take the subsequent filtrate, as the test solution. Determine the hypoxanthine content according to the instrument of Example 2 and the chromatographic conditions under item "1" of Example 2. As shown in Table 25, there is no obvious difference in the data of ultrasonic extraction for 15 minutes, 30 minutes, and 45 minutes, so ultrasonic treatment for 15 minutes is selected as the extraction time for content determination.

[0173] Table 25 Content determination of centipede formula granules at different extraction times

[0174]

[0175] 1.4 Determination of the preparation method of the test solution

[0176] In summary, the final preparation method of the test solution is determined as follows: take the centipede formula granules, grind, take about 0.2g, weigh, put it in a stoppered conical flask, add 25mL of 30% methanol, stopper, weigh, ultrasonically treat (power 400W, frequency 40kHz) for 15 minutes, cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, and take the filtrate to obtain.

[0177] 2. Exclusive inspection

[0178] The auxiliary material of centipede formula granules is maltodextrin. This experiment investigates the effect of maltodextrin on quantitative detection. Take maltodextrin and centipede formula granules and prepare negative solution and test solution according to the method of "4.4" in Example 2, and inject them into liquid chromatograph with hypoxanthine reference solution, and measure according to the instrument of Example 2 and the chromatographic conditions under "1" in Example 2. At the same time, continue to run the same elution time according to the endpoint gradient ratio, examine the chromatographic peak after the specified time, and record the chromatogram. See Fig.16 , Fig.17 .

[0179] The results showed that maltodextrin had no interference with the determination of hypoxanthine content. The separation degree between the hypoxanthine peak and the adjacent chromatographic peak in the chromatogram of the test sample was greater than 1.5, and there were no other chromatographic peaks in the chromatogram of the test sample after 60 minutes. This method was specific for determining the hypoxanthine content in centipede formula granules.

[0180] 3. Peak purity inspection

[0181] Pipette 10 μl of the test solution and inject it into the high performance liquid chromatograph 1. Scan and detect at 230 nm to 270 nm using a diode array detector (PDA) according to the chromatographic conditions under item "1" of Example 2, and calculate the peak purity. Fig.18 The purity angle (0.260) of the hypoxanthine peak in the test sample chromatogram was less than the purity threshold (0.268), and both were less than 1, indicating that the chromatographic peak purity was qualified.

[0182] 4. Linear relationship investigation

[0183] 101.7 μg / mL hypoxanthine reference substance stock solution was measured and graded to dilute reference substance solutions of different concentrations. 10 μl of the solution was injected in sequence according to the chromatographic conditions under item "1" of Example 2, and the chromatographic peak area was recorded. The standard curve was drawn with the peak area as the ordinate (y) and the reference substance concentration as the abscissa (x). The regression equation was obtained as follows: y=41991x+6162.1, and the correlation coefficient was r=0.9999. This indicated that within the range of 1.02~50.85 μg / mL, the reference substance concentration had a good linear relationship with the peak area.

[0184] 5. Precision inspection

[0185] 5.1 Injection precision

[0186] Take hypoxanthine reference solution, repeat injection 6 times according to the chromatographic conditions under item "1" of Example 2, and calculate RSD value by retention time and hypoxanthine peak area. As shown in Table 26, RSD of hypoxanthine retention time is 0.07%, and RSD of peak area is 0.33%, and the instrument precision is good.

[0187] Table 26 Results of precision test of centipede formula granules

[0188]

[0189] 5.2 Repeatability study

[0190] Take the centipede formula granules, grind them, take about 0.2g, weigh them, and prepare 6 copies in parallel. Prepare the test solution according to "4.4" of Example 2, and determine the hypoxanthine content in the test solution under the chromatographic conditions under "1" of Example 2. As shown in Table 27, the average hypoxanthine content is 1.488mg / g, RSD<2%, and the repeatability of the analytical method is good.

[0191] Table 27 Results of repeatability test of centipede formula granule content determination

[0192]

[0193] 5.3 Intermediate Density Investigation

[0194] Select different measurement times, different HPLCs, and different experimenters, take centipede formula granules, grind, take about 0.2g (6 portions in total), weigh, prepare the test solution according to "4.4" of Example 2, and determine the hypoxanthine content in the test solution according to the chromatographic conditions under "1" of Example 2, and compare with the results under the repeatability item. As shown in Table 28, different analysts operated on different dates and different chromatographs, and the hypoxanthine content RSD was <2%, and the intermediate precision of this analytical method was good.

[0195] Table 28 Intermediate precision results of centipede formula granule content determination

[0196]

[0197] Repeatability: Personnel 1, Instrument: High Performance Liquid Chromatograph 1, No.: JS-01-007; Experimental date: 2022 / 05 / 06.

[0198] Intermediate precision: Personnel 2, Instrument: High Performance Liquid Chromatograph 2, No.: JS-01-012; Experimental date: 2022 / 05 / 09.

[0199] 6. Accuracy test

[0200] Weigh 10.23 mg of hypoxanthine reference substance, place in a 100 mL volumetric flask, dissolve and dilute to the mark with 10% methanol to make a solution containing 101.70 μg per mL. Then measure 15 mL to a 250 mL volumetric flask, add 30% methanol to dilute to the mark, and make an accuracy reference solution containing 6.101 μg per mL. Take centipede formula granules, grind, take about 0.1 g, a total of 6 portions, weigh, add 25 mL of the above-mentioned accuracy reference solution respectively, weigh, ultrasonically treat (power 400 W, frequency 40 kHz) for 15 minutes, cool, weigh again, supplement the weight loss with 30% methanol, shake well, filter, and take the filtrate. Determine the hypoxanthine content in the solution according to the chromatographic conditions under "1" of Example 2, and calculate the recovery rate of sample addition. As shown in Table 29, the recovery rate of hypoxanthine is 100.59%~102.22%, the average recovery rate is 101.54%, and the RSD is 0.55%. This method has high accuracy.

[0201] Table 29 Centipede formula granule content determination sample addition recovery results

[0202]

[0203] 7. Stability test

[0204] Take the centipede formula granules, grind, take about 0.2g, weigh, prepare the test solution according to Example 2 "4.4", and measure the hypoxanthine peak area at 0, 10, 30, 40, and 52 hours according to the chromatographic conditions under Example 2 "1", and calculate the peak area RSD. As shown in Table 30, the RSD of the hypoxanthine peak area is less than 2%, and the test solution is stable and measurable within 52 hours.

[0205] Table 30 Centipede formula granules content determination test sample stability investigation results

[0206]

[0207] 8. Durability inspection

[0208] 8.1 Investigation of different column temperatures

[0209] Take centipede formula granules, grind, take about 0.2g, weigh, prepare test solution according to Example 2 "4.4", except column temperature is 28 ℃, 30 ℃, 32 ℃, the rest is measured according to the chromatographic conditions under Example 2 "1", and compare the influence of different column temperatures on the determination of hypoxanthine content. As shown in Table 31, the RSD of hypoxanthine content under three different column temperatures is 4.00%, and the column temperature setting of 30 ℃ for this analysis method meets the requirements.

[0210] Table 31 Results of durability study on centipede formula granule content determination at different column temperatures

[0211]

[0212] 8.2 Investigation of different flow rates

[0213] Compare the effects of different flow rates of 0.9mL / min, 1.0mL / min, and 1.1mL / min on the determination of hypoxanthine content. Take the test solution (different column temperatures) in the "durability study" of this embodiment, and determine the content of hypoxanthine according to the chromatographic conditions under item "1" of Example 2 except the flow rate. As shown in Table 32, the RSD of hypoxanthine content at three different flow rates is 4.08%, and the flow rate setting of this analytical method at 1.0mL / min meets the requirements.

[0214] Table 32 Centipede formula granule content determination different flow rate durability investigation results

[0215]

[0216] 8.3 Investigation of different chromatographic columns

[0217] Compare the effects of chromatographic columns 1, 2, and 3 on the content determination of centipede formula granules. Take centipede formula granules, grind, take about 0.2g, weigh, prepare the test solution according to "4.4" of Example 2, and determine the hypoxanthine content according to the chromatographic conditions under "1" of Example 2. As shown in Table 33, the RSD of the hypoxanthine content in the three chromatographic columns is 1.86%, and the RSD value is <2%. This method has good durability for the three chromatographic columns.

[0218] Table 33 Results of different chromatographic columns for determination of centipede formula granules

[0219]

[0220] 9. Summary

[0221] The methodological investigation of the quantitative determination of hypoxanthine in centipede formula granules showed that the blank of this chromatographic condition had no interference and good specificity; the theoretical plate number was greater than 10,000, and the linearity of hypoxanthine in the concentration range of 1.02-50.85 μg / mL was good; the injection precision, repeatability, and intermediate precision were good; the test solution was stable within 52 hours at room temperature; the recovery rate was between 100.59% and 102.22%, with high accuracy; the column temperature was 30°C and the flow rate was 1.0 mL / min, with good durability. It has good durability for different chromatographic columns. This method can be accurately used for the quantitative determination of hypoxanthine in centipede formula granules.

Claims

1. A liquid chromatography characteristic spectrum qualitative detection method for centipede formula granules, characterized in that: The preparation method of the centipede formula granules comprises the following steps: taking 3000g of centipede pieces, decocting with water, filtering, concentrating the filtrate into a clear paste, adding auxiliary material maltodextrin, drying, mixing, granulating, and making 1000g, that is, 1g of centipede formula granules is equivalent to 3g of pieces; The liquid chromatography characteristic spectrum qualitative detection method of the centipede formula granules comprises the following steps: Preparation of test solution: Take centipede formula granules, use 10%~50% methanol aqueous solution for ultrasonic or heating reflux extraction, filter, and take the filtrate as the test solution; Preparation of reference solution: Take hypoxanthine reference, uracil reference, uridine reference and guanosine reference, and dissolve them in methanol-water solution to prepare reference solution; Injection test: Take the test solution and the reference solution for injection respectively; the chromatographic column is filled with octadecylsilane bonded silica gel, and the chromatographic column parameters are: column length 250mm, inner diameter 4.6mm, particle size 5μm; methanol is used as mobile phase A, 0.1% glacial acetic acid solution is used as mobile phase B, and gradient elution is performed; the flow rate is 0.9mL~1.1mL per minute, the column temperature is 28℃~32℃, and the detection wavelength is 248nm; the elution program includes the following procedures: 0~15 minutes: the volume percentage of mobile phase A is 0%; 15 minutes~30 minutes: the volume percentage of mobile phase A increases from 0% to 10%; 30~45 minutes: the volume percentage of mobile phase A increases from 10% to 40%; Chromatographic determination: 9 characteristic peaks should be present in the chromatogram of the test sample, and peaks 1, 4, 6, and 8 correspond to the retention times of the chromatographic peaks of uracil, hypoxanthine, uridine, and guanosine; the peak corresponding to hypoxanthine, i.e. peak 4, is taken as the S1 peak, and the relative retention times of peaks 2, 3, and 5 with the S1 peak are calculated, and the relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.66 for peak 2, 0.90 for peak 3, and 1.24 for peak 5; the peak corresponding to guanosine, i.e. peak 8, is taken as the S2 peak, and the relative retention times of peaks 7, 9, and S2 peak are calculated, and the relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.97 for peak 7, and 1.29 for peak 9.

2. The liquid chromatography characteristic spectrum qualitative detection method of a centipede formula granule according to claim 1, characterized in that: In the test solution preparation step, the extraction time is 15 minutes to 45 minutes.

3. The liquid chromatography characteristic spectrum qualitative detection method of a centipede formula granule according to claim 1, characterized in that: In the test solution preparation step, the extraction method is ultrasonic extraction; the ultrasonic power is 400W and the frequency is 40kHz.

4. The liquid chromatography characteristic spectrum qualitative detection method of a centipede formula granule according to claim 1, characterized in that: In the reference solution preparation step, the concentration of the methanol aqueous solution is 10%.

5. The liquid chromatography characteristic spectrum qualitative detection method of a centipede formula granule according to claim 4, characterized in that: In the reference solution preparation step, the concentrations of hypoxanthine, uracil, uridine and guanosine in the reference solution are 10 μg, 5 μg, 8 μg and 8 μg per mL respectively.

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