CN118050451A - Liquid chromatography qualitative and quantitative detection method for centipede formula particles - Google Patents

Abstract

The invention belongs to the technical field of liquid chromatography analysis of traditional Chinese medicine formula particles, and particularly relates to a liquid chromatography qualitative and quantitative detection method of centipede formula particles. The liquid chromatography qualitative and quantitative detection method of the centipede formula particles adopts 10% -50% methanol aqueous solution as an extraction solvent, and adopts ultrasonic or heating reflux extraction, wherein the selected reference substances comprise hypoxanthine, uracil, uridine and guanosine; the chromatographic column filler is chromatographic column, octadecylsilane chemically bonded silica is used as filler, methanol and 0.1% glacial acetic acid solution are used as mobile phase, and the detection wavelength is 248nm. The method can show 9 characteristic peaks within 40 minutes of elution time, is suitable for qualitative and quantitative detection, has good adaptability to chromatographic columns and the like, has good specificity, stability, quality control capability and analysis efficiency, is suitable for quality control of one measurement and multiple evaluation, and is easy to use and convenient to popularize.

Description

Liquid chromatography qualitative and quantitative detection method for centipede formula particles

Technical Field

The invention belongs to the technical field of liquid chromatography analysis of traditional Chinese medicine formula particles, and particularly relates to a liquid chromatography qualitative and quantitative detection method of centipede formula particles.

Background

Centipede belongs to common animal traditional Chinese medicine materials, is pungent, warm and toxic. Has the effects of stopping endogenous wind, relieving spasm, dredging collaterals, relieving pain, eliminating toxic substances and resolving hard mass. Can be used for treating internal movement of liver wind, convulsion, infantile convulsion, apoplexy, hemiplegia, tetanus, rheumatism, migraine, sore, scrofula, and snake and insect bite. The Chinese pharmacopoeia 2020 edition specifies that the centipede base source is a dried body of Scolopendra subspinipes (Scolopendra) Scolopendra subspinipes (Koch).

The traditional Chinese medicine formula granule is prepared by adopting a water extraction process for single traditional Chinese medicine decoction pieces, retains the traditional medicine characteristics, has the advantages of convenient carrying and easy brewing, and reduces the medicine decocting burden of patients and medical institutions. Along with the ending of the test points in China in 2021, the traditional Chinese medicine formula granules are widely popularized in clinic, and the use amount of the traditional Chinese medicine formula granules is over that of traditional Chinese medicine decoction pieces. However, part of traditional Chinese medicine formula particles, especially traditional Chinese medicine formula particles of animal medicines, lack of corresponding quality standards and chromatographic identification methods, so that the quality control difficulty is high. For centipede formula particles, the influence of undefined medicinal components and the like exists, and the effective chromatographic analysis means for performing quality control such as one-measurement-multiple-evaluation of the centipede formula particles is lacking in the pharmacopoeia of China at present.

Domestic scholars and enterprises have conducted research and study on this, for example, xu Yuansheng and the like have developed a specific PCR identification method for centipede formula particles (Xu Yuansheng and the like. Site-specific PCR identification of centipede formula particles. Journal of Chinese laboratory prescriptions, 2024, 30 (4): 48-54.). The gene amplification equipment and technology required by the method are not promoted in the medicine enterprises, and lack of promotion foundation. CN202111338155.2 provides an HPLC chromatographic detection method for centipede medicinal materials, the target is hypoxanthine, xanthine and quinolines in the medicinal materials, the mobile phase is acetonitrile-ammonium phosphate system, 5 substances can be detected and analyzed at the same time, but most quinolines are not proved to exist in centipede formula particles. CN202310937609.0 provides an ultra-high performance liquid chromatography (UPLC) detection method for centipede formula particles, a mobile phase is a methanol-potassium dihydrogen phosphate system, and 6 characteristic peaks can be simultaneously presented, wherein 3, 8-dihydroxyquinoline is identified, the retention time of the characteristic peaks exceeds 46 minutes, the time consumption is long, the detection efficiency is low, the characteristic peaks are few, and the quality control capability is weak. The invention adopts a C18 reverse phase column to compare various elution systems, but finally the methanol-acetic acid elution system is abandoned due to factors such as the separation degree, the baseline stability, the peak shape and the like, and the chromatographic peak obtained by the methanol-acetic acid elution system in the three elution systems is the least. CN202310937552.4 provides a High Performance Liquid Chromatography (HPLC) detection method of centipede formula particles, the mobile phase is a methanol-water system, 8 characteristic peaks can be presented, and uracil, hypoxanthine, xanthine, inosine, guanosine and part of amino acids are involved. And the retention time of 8 characteristic peaks is within 40 minutes, so that the analysis efficiency and the quality control capability are improved. However, the chromatographic column of the method has poor adaptability, and only DIKMAPLATISIL ODS C columns meet the requirements.

So far, reports on component analysis and quality control means of centipede formula particles are still few, and development of liquid chromatography analysis means of centipede formula particles is restricted.

Disclosure of Invention

In order to solve the problems, the inventor of the invention unexpectedly finds that in the process of executing a subject research task and a quality standard research, a C18 chromatographic column is adopted as a stationary phase, a methanol-glacial acetic acid system is adopted as a mobile phase, the detection wavelength is 248nm, liquid chromatography detection is carried out on centipede reference medicinal materials and centipede formula particles, 9 characteristic peaks can be presented in 40 minutes of elution time, the peak shape and the separation level baseline stability are good, the method is suitable for various C18 columns, the method specificity, the stability and the precision are good, no gene amplification equipment is required, and the method is suitable for popularization and application in enterprises, and the liquid chromatography qualitative detection, quantitative detection method and research and development materials are reported to the authorities for rechecking and recording.

Therefore, the invention provides a liquid chromatography qualitative and quantitative detection method for centipede formula particles.

Wherein the Scolopendra is Scolopendra (Scolopendra) based on Scolopendra (Scolopendra family animal Scolopendra acutangula (Scolopendra) Scolopendra subspinipes mutilans L. Koch in the year 2020 edition of Chinese pharmacopoeia). The Scolopendra granule is prepared by processing Scolopendra Scolopendra subspinipesmetans L.Koch dried body, decocting with water, concentrating, and adding maltodextrin.

The liquid chromatography qualitative detection method of the centipede formula particles comprises the following steps:

preparing a test solution: taking centipede formula particles, carrying out ultrasonic or heating reflux extraction by adopting a methanol aqueous solution with the concentration of 10% -50%, filtering, and taking a subsequent filtrate as a sample solution;

preparing a reference substance solution: dissolving hypoxanthine reference substance, uracil reference substance, uridine reference substance and guanosine reference substance in methanol water solution to obtain reference substance solution;

And (3) sample injection detection: taking sample solutions of the sample and the reference substance respectively; the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler, and the parameters of the chromatographic column are as follows: the column length is 250mm, the inner diameter is 4.6mm, and the grain diameter is 5 μm; methanol is used as a mobile phase A, and 0.1% glacial acetic acid solution is used as a mobile phase B, and gradient elution is carried out; the flow rate is 0.9-1.1 mL/min, the column temperature is 28-32 ℃, and the detection wavelength is 248nm; the elution procedure included the following: 0-15 minutes: the volume percentage of the mobile phase A is 0%;15 minutes to 30 minutes: the volume percent ratio of the mobile phase A is increased from 0% to 10%; 30-45 minutes: the volume percentage of the mobile phase A is increased from 10% to 40%.

Preferably, in the step of preparing the sample solution, the extraction time is 15 minutes to 45 minutes.

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

Preferably, in the reference solution preparation step, the concentration of the methanol aqueous solution is 10% -30%.

Preferably, in the step of preparing the reference solution, the concentration of hypoxanthine, uracil, uridine and guanosine in the reference solution is 10 mug, 5 mug, 8 mug and 8 mug per mL in sequence.

Preferably, in the step of preparing the sample solution, the extraction solvent is a 30% aqueous methanol solution.

Preferably, the qualitative detection method further comprises the steps of:

Qualitative identification of centipede formula particles: and (3) analyzing the chromatograms obtained in the sample injection detection step, wherein the chromatograms of the samples to be tested show 9 characteristic peaks, and peak 1, peak 4, peak 6 and peak 8 correspond to retention time of chromatograms of uracil, hypoxanthine, uridine and guanosine. Peak 4, which corresponds to the hypoxanthine reference peak, was used as the S1 peak, and the relative retention times of peak 2, peak 3, peak 5 and S1 peak were calculated to be within ±10% of the predetermined values: 0.66 (Peak 2), 0.90 (Peak 3), 1.24 (Peak 5). Peak 8, which is the peak corresponding to the guanosine control reference peak, was used as the S2 peak, and the relative retention time of peak 7, peak 9 and S2 peak was calculated to be within ±10% of the prescribed value, which is: 0.97 (peak 7), 1.29 (peak 9).

The liquid chromatography quantitative detection method of the centipede formula particles comprises the following steps:

preparing a test solution: taking centipede formula particles, carrying out ultrasonic or heating reflux extraction by adopting a methanol aqueous solution with the concentration of 10% -50%, filtering, and taking a subsequent filtrate as a sample solution;

Preparing a reference substance solution: dissolving hypoxanthine reference substance in methanol water solution to obtain hypoxanthine reference substance solution;

And (3) sample injection detection: taking sample solutions of the sample and the hypoxanthine reference substance, respectively injecting samples, and measuring the hypoxanthine content in the sample solutions; the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler, and the parameters of the chromatographic column are as follows: the column length is 250mm, the inner diameter is 4.6mm, and the grain diameter is 5 μm; methanol is used as a mobile phase A, and 0.1% glacial acetic acid solution is used as a mobile phase B, and gradient elution is carried out; the flow rate is 1.0 mL/min, the column temperature is 30 ℃, and the detection wavelength is 248nm; the elution procedure included the following: 0-15 minutes: the volume percentage of the mobile phase A is 0%;15 minutes to 30 minutes: the volume percent ratio of the mobile phase A is increased from 0% to 10%; 30-45 minutes: the volume percentage of the mobile phase A is increased from 10% to 40%.

Preferably, in the step of preparing the sample solution, the extraction time is 15 minutes to 45 minutes.

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

Preferably, in the reference solution preparation step, the concentration of the methanol aqueous solution is 10% -30%.

Preferably, the concentration of hypoxanthine in the hypoxanthine control solution is 10 μg per mL.

Preferably, in the step of preparing the sample solution, the extraction solvent is a 30% aqueous methanol solution.

The solubility and solubility of uracil, hypoxanthine, uridine, guanosine are common knowledge in the art, and a person skilled in the art can select different aqueous solutions of methanol 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 adopts 30% methanol as a solvent, and the concentration of the reference substance in the solution is 0.1mg/mL.

Advantageous effects

(1) The chromatographic column of the liquid chromatography detection method has good adaptability: the analysis requirements are met by both the JADE-PAK ODS-AQ (4.6mm.times.250 mm,5 μm) and COSMOSIL Packed Column C18-MS-II (4.6mm.times.250 mm,5 μm) types of C18 columns;

(2) The liquid chromatography detection method has good adaptability to instruments and equipment: the analysis requirements of the Waters2695 and UltiMate and 3000 high performance liquid chromatographs are met;

(3) The liquid chromatography detection method has good adaptability to the extraction conditions: ultrasonic or heating reflux extraction is carried out on a sample by adopting a methanol aqueous solution with the concentration of 10% -50% for 15-45 minutes, so that a chromatogram meeting the analysis requirement can be obtained;

(4) The liquid chromatography detection method has good quality control capability and analysis efficiency: 9 characteristic peaks can be obtained within 40 minutes of elution time, so that the method has higher quality control capability and higher analysis efficiency;

(5) The liquid chromatography detection method has the characteristics of good separation degree of characteristic peaks, good peak shape, stable base line, qualitative and quantitative discrimination capability, and is suitable for one-measurement-multiple-evaluation.

Drawings

FIG. 1 is a graph comparing the characteristic patterns of two extraction modes of Scolopendra formulation granule in example 2;

FIG. 2 is a graph showing comparison of characteristic patterns of different extraction solvents of Scolopendra formulation granule in example 2;

FIG. 3 is a graph showing comparison of characteristic patterns of Scolopendra formulation granules of example 2 at different extraction times;

FIG. 4 is a characteristic spectrum-specific investigation spectrum of Scolopendra formulation granule in example 3;

FIG. 5 is a graph of example 3 showing a two-fold run time profile of a specificity study of a characteristic spectrum of a centipede formula particle;

FIG. 6 is a chart of sample injection precision investigation stack of the centipede formula particle characteristic spectrum in example 3;

FIG. 7 is a graph of the repeat investigation stack of the characteristic spectrum of the centipede formula particle in example 3;

FIG. 8 is a chart of a stack of intermediate precision studies of the characterization of the Scolopendra formulation granules in example 3;

FIG. 9 is a stack of stability studies for the characterization of the particles of the centipede formulation of example 3;

FIG. 10 is a chart showing the stacking of different column temperature studies of the characteristic diagram of Scolopendra formulation particles in example 3;

FIG. 11 is a chart showing the flow rate of the centipede formula particle characteristic diagram in example 3;

FIG. 12 is a chart of the characteristics of the centipede formula particles of example 3, stacked in FIG. 1 without chromatographic column inspection;

FIG. 13 is a chart of the characteristics of the Scolopendra formulation particles of example 3, stacked with no chromatographic column;

FIG. 14 is a graph showing a stacking of the characteristic patterns of 3 batches of Scolopendra formula granules in example 4;

FIG. 15 is a graph of the comparative characteristics of Scolopendra formula granules in example 4;

FIG. 16 is a plot of a specific study of the content of particles in the Scolopendra formulation in example 5;

FIG. 17 is a graph of the two-fold run time plot of the specificity study for the centipede formula particle content determination in example 5;

FIG. 18 is a chart depicting peak purity measurements for the content of particles of the centipede formulation of example 5;

in the figure, the chromatogram peak 1 is uracil, peak 4 is hypoxanthine, peak 6 is uridine, and peak 8 is guanosine.

Detailed Description

The method for identifying the centipede formula particles according to the invention is described below by means of specific examples. The embodiments are illustrative examples and are not to be construed as limiting the scope of protection. In the examples, the methanol concentration is a volume ratio, i.e., a volume percentage concentration, and for example, 10%, 30%, 50% methanol as the extraction solvent is a 10%, 30%, 50% aqueous methanol solution, respectively, unless otherwise specified. Unless otherwise specified, the glacial acetic acid concentration in the examples is a volume percent concentration, for example, a 0.1% glacial acetic acid solution as mobile phase B is a 0.1% aqueous glacial acetic acid solution by volume. In the examples, RAD is the relative mean deviation and RSD is the relative standard deviation unless otherwise specified; SD is standard deviation. The information of the instrument, the reagent and the like used in the embodiment of the invention is as follows:

Chromatographic column: chromatographic column 1: JADE-PAK ODS-AQ 4.6mm.times.250 mm,5 μm (lot number: US2237A2UE 93); chromatographic column 2: JADE-PAK ODS-AQ 4.6mm.times.250 mm,5 μm (lot number: US2137AJUD 73); chromatographic column 3: COSMOSIL Packed Column 5C 18-MS-II 4.6mm.times.250 mm,5 μm (lot number: manf No K66594).

Reagent: methanol is chromatographic pure and is produced by Colon chemical Co., ltd; glacial acetic acid is chromatographic pure and manufactured by Colon chemical Co., ltd; the water is purified water prepared by an ultrapure water machine.

Reagent: hypoxanthine reference (lot number: 140661-202005, purity: 99.4%, national institute of food and drug testing); uracil control (lot number: PS020117, purity: 98.0%, chengdu Poisson Biotechnology Co., ltd.); uridine (lot number: PS010290, purity: 98.0%, chengdu Poisson Biotechnology Co., ltd.); guanosine (lot number: PS010291, purity: 98.0%, chengdu Poisson Biotechnology Co., ltd.); centipede control (lot number: DST210911-013, cheng Du Le Mei Tian medical science and technology Co., ltd.); centipede formula particles prepared by the applicant and the preparation method comprises the following steps: 3000g of centipede decoction pieces are taken, water is added for decoction, filtration is carried out, the filtrate is concentrated into clear paste (the paste yield of the dry extract is 17.0% -31.0%), auxiliary material maltodextrin is added, drying (or drying and crushing) is carried out, and the mixture is evenly mixed, granulated and prepared into 1000g, thus obtaining 1g of centipede formula particles which are equivalent to 3g of decoction pieces.

Instrument apparatus: high performance liquid chromatograph 1 (Waters 2695, waters technologies limited); high performance liquid chromatograph 2 (UltiMate 3000,3000, zemoer feishi technologies (china) limited); one hundredth analytical balance (CN-LQC 10002, kunshan, kovicat electronics limited); one ten thousandth analytical balance (FA 1004, force science, inc.); an analytical balance (AUW 220D, manufactured by Shimadzu corporation); numerical control ultrasonic cleaner (KH-500 DE, kunshan He Chuang ultrasonic instruments Co., ltd.); centrifuge (TG-16B, a company of Instrument Co., ltd.); ultrapure water system (EKUP-II-10 p, sichuan pure water equipment Co., ltd.); electronic temperature-regulating electric jacket (DZTW, a technology company of Fu Zhou instruments, hangzhou).

The purpose of the individual formulation of the hypoxanthine control solutions in the examples below is to facilitate a quote between the methods of example 2 and example 5, and to facilitate an individual comparison of methodology considerations. In the liquid chromatography qualitative detection operation of Scolopendra granule, hypoxanthine, uracil, uridine and guanosine can be mixed in the same solution as reference solution.

Example 1 preparation of Scolopendra formulation granules

The preparation of the centipede formula particle comprises the following steps:

Processing: taking centipede medicinal materials, removing bamboo chips, cleaning, baking with slow fire to yellow, and cutting.

Extracting: weighing centipede decoction pieces, adding water for decoction twice, adding 8 times of water (w/w) for the first time, and filtering after decoction for 1 hour; adding 6 times of water (w/w) for the second time, decocting for 0.5 hr, filtering, and mixing filtrates.

Concentrating: concentrating the filtrate at 80 deg.c under reduced pressure to obtain fluid extract with relative density of 1.01-1.06 (80 deg.c).

Vacuum drying: taking the fluid extract, adding maltodextrin, vacuum drying (the temperature of a water tank is 80-90 ℃ and the temperature of the inside of the tank is 40-60 ℃), crushing, collecting powder, and sieving to obtain dry paste powder.

Total mixing: and (3) placing the dry paste powder and the calculated amount of sieved maltodextrin into a mixer, and uniformly mixing to obtain the total mixed powder. Maltodextrin calculated amount = decoction piece dosage amount/3-dry paste powder amount

Dry granulating: and (3) taking the total mixed powder, granulating by a dry method, and finishing to obtain granules. The 1g of the formula granule is equivalent to 3g of decoction pieces.

Example 2 extraction Condition investigation of qualitative detection

Instrument apparatus: high performance liquid chromatograph 1.

1. Chromatographic conditions

Octadecylsilane chemically bonded silica is used as a filler (chromatographic column 1); eluting with methanol as mobile phase A and 0.1% glacial acetic acid solution as mobile phase B according to the procedure of Table 1; the flow rate is 1.0mL per minute; the column temperature is 30 ℃; the detection wavelength is 248nm, and the sample injection amount is 10 mu L. The theoretical plate number is not less than 10000 calculated according to the hypoxanthine peak.

TABLE 1 gradient elution procedure

2. Preparation of reference solutions

2.1 Preparation of control drug reference solution: taking 0.2g of centipede reference medicine, placing the centipede reference medicine into a conical flask with a plug, adding 25mL of 30% methanol, weighing, heating and refluxing for 15 minutes, cooling, weighing again, supplementing the reduced weight with 30% methanol, shaking uniformly, taking supernatant, filtering, and taking subsequent filtrate as reference substance solution of the reference medicine.

2.2 Preparation of hypoxanthine control solution: weighing hypoxanthine reference substance, and adding 10% methanol to obtain solution containing 10 μg per mL.

2.3 Preparation of uracil, uridine, guanosine reference substance mixed solution: taking uracil reference substance, uridine reference substance and guanosine reference substance, weighing, adding 10% methanol to prepare mixed solution containing uracil 5 μg, uridine 8 μg and guanosine 8 μg per mL, and taking as reference substance mixed solution.

3. Determination and chromatographic qualitative identification

Respectively sucking reference solution (i.e. reference solution of reference material, hypoxanthine reference solution and reference mixed solution of reference material) and sample solution (10 μl), respectively, and measuring with liquid chromatograph.

And (3) chromatographic qualitative identification: according to the detection method, the chromatogram of the test sample should show 9 characteristic peaks corresponding to the retention time of 9 characteristic peaks in the chromatogram of the reference material of the control medicine. Wherein peak 1, peak 4, peak 6, peak 8 correspond to uracil, hypoxanthine, uridine, guanosine, respectively, chromatographic peak retention times. Calculating the relative retention time of peak 2, peak 3, peak 5 and S1 peak by taking peak 4 corresponding to the hypoxanthine reference peak as S1 peak, wherein the relative retention time is within +/-10% of the specified value, and the specified value of the relative retention time is: 0.66 (Peak 2), 0.90 (Peak 3), 1.24 (Peak 5). Calculating the relative retention time of peak 7, peak 9 and S2 peak by taking peak 8 corresponding to the guanosine reference peak as S2 peak, wherein the relative retention time is within + -10% of the specified value, and the specified value of the relative retention time is: 0.97 (peak 7), 1.29 (peak 9).

4. Pretreatment method for test article

4.1 Investigation of extraction method

Taking centipede formula particles, grinding, taking about 0.2g (two groups), weighing, placing into a conical bottle with a plug, adding 25mL of 10% methanol, sealing, weighing, respectively carrying out ultrasonic treatment (power 400W and frequency 40 kHz) or heating and refluxing for 30 minutes, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, filtering, and taking subsequent filtrate. The peak shape and the separation degree of the 9 characteristic peaks are analyzed, and the influence of the total peak area/sample weighing of the 9 characteristic peaks on the chromatogram by different extraction modes is calculated. The results are shown in Table 2 and FIG. 1. The results show that: there was no significant difference between the ultrasonic and reflux extracted chromatographic peaks and the "total peak area/sample size". Ultrasonic extraction is convenient, so ultrasonic treatment is selected as an extraction mode.

Table 2 investigation of different extraction modes of the characteristic spectrum of Scolopendra formulation particles

4.2 Extraction solvent investigation

The pre-experiment shows that the methanol extraction effect is superior to that of ethanol, and the experiment examines the extraction effects of methanol with different concentrations.

Taking centipede formula particles, grinding, taking about 0.2g (three groups), weighing, placing into a conical flask with a plug, respectively adding 25mL of 10% methanol, 30% methanol and 50% methanol, weighing, performing ultrasonic treatment (power 400W and frequency 40 kHz) for 30 minutes, cooling, weighing again, respectively adding corresponding solvents to supplement weight loss, shaking uniformly, filtering, and taking subsequent filtrate. The peak shape and the degree of separation of the 9 characteristic peaks were analyzed, and the "total peak area/sample amount" of the 9 characteristic peaks was calculated to compare the influence of different extraction solvents on the chromatogram. The results are shown in Table 3 and FIG. 2. The results show that: there was no significant difference between the chromatographic peaks and the "total peak area/sample size" of the different solvent extractions, and 30% methanol was selected as the extraction solvent in combination with the method of example 5.

Table 3 extraction solvent investigation of centipede formula particle characteristic pattern

4.3 Extraction time investigation

Taking centipede formula particles, grinding, taking about 0.2g (three groups), weighing, placing into a conical bottle with a plug, adding 25mL of 30% methanol, sealing, weighing, respectively carrying out ultrasonic treatment (power 400W and frequency 40 kHz) for 15 minutes, 30 minutes and 45 minutes, cooling, supplementing the lost weight with 30% methanol, shaking uniformly, filtering, and taking the subsequent filtrate. The peak shape and the degree of separation of the 9 characteristic peaks were analyzed, and the effect of the "total peak area/sample amount" of the 9 characteristic peaks on the chromatogram was calculated, compared with the different extraction times. The results are shown in Table 4 and FIG. 3. The results show that: there was no significant difference in the chromatographic peaks and the "total peak area/sample size" extracted at different extraction times. 15 minutes was chosen as the extraction time.

Table 4 investigation of different extraction times of the characteristic spectrum of Scolopendra formulation particles

4.4 Determination of the method for preparing the sample solution

As shown in fig. 1 to 3, the retention times (minutes) of the peaks 1 to 9 are respectively in this order 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、38.65~39.23.

In summary, the preparation method of the test sample solution is determined as follows: taking centipede formula particles, grinding, taking about 0.2g, weighing, placing into a conical flask with a plug, adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 15 minutes, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

Example 3 methodological verification of qualitative detection

1. Investigation of specificity

The auxiliary material of the centipede formula granule is maltodextrin. The experiment examined the effect of maltodextrin on qualitative detection. A negative sample solution was prepared from maltodextrin as described in example 2 under "4.4" item. Sample analysis was performed on the sample solution, the negative sample solution, and the reference solution according to the apparatus of example 2 and the chromatographic conditions under the conditions of example 2"1", and a chromatogram was recorded. And simultaneously, continuously running the same elution time according to the end point gradient proportion, examining the chromatographic peak condition after the specified time, and recording a chromatogram. The results are shown in FIG. 4 and FIG. 5, respectively. The results show that the test sample shows 9 characteristic peaks corresponding to the retention time of 9 characteristic peaks of the control medicinal material, wherein peak 1, peak 4, peak 6 and peak 8 correspond to the retention time of uracil, hypoxanthine, uridine and guanosine chromatographic peaks. Maltodextrin has no interference to the chromatogram, and no other chromatographic peaks exist in the test sample after 60 minutes. The method has specificity.

2. Precision investigation

2.1 Precision of sample injection

Taking centipede formula particles, grinding, taking about 0.2g, weighing, preparing a test sample solution according to the method of 4.4 of example 2, carrying out sample injection analysis according to the instrument of example 2 and the chromatographic conditions under the condition of 2"1 of example 2"1, continuously injecting the same sample solution for 6 times, taking the peak corresponding to the peak of the hypoxanthine reference substance, namely the peak 4 as an S1 peak, and calculating the relative retention time of the peak 2, the peak 3, the peak 5 and the S1 peak; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; and calculating the relative peak areas of the peaks 1-3, 5-9 and S1, and calculating the RSD value. The results are shown in Table 5, table 6 and FIG. 6. The result shows that the relative retention time RSD range of the peak 2, the peak 3, the peak 5 and the S1 peak is 0.04% -0.12%; the relative retention time RSD value of the peak 7, the peak 9 and the S2 peak is 0.00% -0.09%; the relative peak area RSD values of the peaks 1-3, 5-9 and S1 are 0.38-2.25%; RSD values are all less than 5%, and the precision of the instrument is good.

Table 5 results table (relative retention time) for investigating precision of centipede formula particle characteristic spectrum

Table 6 results table (relative peak area) of the centipede formula particle feature map precision investigation

2.2 Repeatability investigation

Taking centipede formula particles, grinding, taking about 0.2g, weighing in parallel 6 parts, preparing a sample solution according to the method of example 2 ' 4.4 ', carrying out sample injection analysis according to the chromatographic conditions under the instrument of example 2 and the method of example 2"1 ', taking the peak corresponding to the peak of the hypoxanthine reference substance, namely the peak 4 as an S1 peak, and calculating the relative retention time of the peak 2, the peak 3, the peak 5 and the S1 peak; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; and calculating the relative peak areas of the peaks 1-3, 5-9 and S1, and calculating the RSD value. The results are shown in Table 7, table 8 and FIG. 7. The result shows that the relative retention time RSD range of the peak 2, the peak 3, the peak 5 and the S1 peak is 0.03% -0.64%; the relative retention time RSD value of the peak 7, the peak 9 and the S2 peak is 0.00% -0.17%; the relative retention time RSD values are less than 5%, and the relative peak area RSD values of the peaks 1-3, 5-9 and S1 are 0.31% -2.67%; the relative peak area RSD values are all less than 5%, and the qualitative method has better repeatability.

TABLE 7 Scolopendra formulation granule characteristics pattern repeatability test results table (relative retention time)

Table 8 Scolopendra formulation granule characteristic pattern repeatability test results table (relative peak area)

2.3 Intermediate precision investigation

Operating on different instruments at different times by different analysts, taking centipede formula particles, grinding, taking about 0.2g, weighing in parallel 6 parts, preparing a sample solution according to the method of example 2 ' 4.4 ', carrying out sample injection analysis according to the chromatographic conditions under the item of example 2"1 ', taking the peak corresponding to the peak of the hypoxanthine reference substance, namely the peak 4 as an S1 peak, and calculating the relative retention time of the peak 2, the peak 3, the peak 5 and the S1 peak; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; and calculating the relative peak areas of the peaks 1-3, 5-9 and S1, and calculating the RSD value. The results are shown in Table 9, table 10 and FIG. 8. Experimental results show that the relative retention time RSD of the peak 2, the peak 3, the peak 5 and the peak S1 ranges from 0.03% to 1.52% when the personnel B and the personnel A operate on different instruments at different times; the relative retention time RSD value of the peak 7, the peak 9 and the S2 peak is 0.08% -0.64%; relative retention time RSD values were all < 5%. The relative peak area RSD values of the peaks 1-3, 5-9 and S1 are 1.87% -7.49%; the relative peak area RSD values of the peak 1 and the peak 3 are more than 5 percent. The qualitative method has good intermediate precision relative to the retention time.

Table 9 intermediate precision examination results table (relative retention time) for Scolopendra formula granules

Table 10 intermediate precision examination results table (relative peak area) for centipede formula particles

In tables 9 and 10, repeatability: personnel a, instrument: high performance liquid chromatograph 1, numbered: JS-01-007; experimental date: 2022/05/06; intermediate precision: personnel B, instrument: high performance liquid chromatograph 2, numbered: JS-01-012; experimental date: 2022/05/09.

3. Stability investigation

Taking centipede formula particles, grinding, taking about 0.2g, weighing, preparing a test solution according to the example 2 ' 4.4 ', carrying out sample injection analysis according to the instrument of the example 2 and the chromatographic conditions under the example 2"1 ', carrying out sample injection analysis at the 0 th, 10 th, 30 th, 40 th and 52 th hours respectively, taking the peak corresponding to the hypoxanthine reference peak, namely the peak 4 as an S1 peak, and calculating the relative retention time of the peak 2, the peak 3, the peak 5 and the S1 peak; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; and calculating the relative peak areas of the peaks 1-3, 5-9 and S1, and calculating the RSD value. The results are shown in Table 11, table 12 and FIG. 9. The result shows that the relative retention time RSD range of the peak 2, the peak 3, the peak 5 and the S1 peak is 0.09% -0.26%; the relative retention time RSD value of the peak 7, the peak 9 and the S2 peak is 0.00% -0.33%; the relative peak area RSD values of the peaks 1-3, 5-9 and S1 are 0.54% -3.18%; the relative retention time and relative peak area RSD were both < 5%, indicating that the test solution was stable and measurable when left for 52 hours.

Table 11 stability test results Table (relative retention time) of Scolopendra formula granule characteristics

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Table 12 stability test results Table (relative peak area) of Scolopendra formula granule characteristics map

4. Durability inspection

4.1 Different column temperature investigation

The effect of different column temperatures, 28 ℃,30 ℃ and 32 ℃ on the chromatograms was compared. A test solution was prepared as in example 2"4.4" by grinding the centipede formula particles to about 0.2 g. Other chromatographic conditions except column temperature are the same as the instrument of example 2 and the chromatographic conditions of example 2"1", sample injection analysis is carried out, the peak corresponding to the hypoxanthine reference peak, namely peak 4 is the S1 peak, and the relative retention time of peak 2, peak 3, peak 5 and S1 peak is calculated; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; and calculating the relative peak areas of the peaks 1-3, 5-9 and S1, and calculating the RSD value. The results are shown in Table 13, table 11 and FIG. 10. The result shows that the relative retention time RSD range of the peak 2, the peak 3, the peak 5 and the S1 peak is 0.33% -0.80%; the relative retention time RSD value of the peak 7, the peak 9 and the S2 peak is 0.00% -1.82%; the relative peak area RSD values of the peaks 1-3, 5-9 and S1 are 1.14% -5.34%; the relative retention time RSD is less than 5%, and the relative peak area RSD of peak 8 is more than 5%, which shows that the qualitative method has better relative retention time durability when the column temperature is within + -2 ℃.

Table 13 Scolopendra formulation granule characteristic spectrum column temperature investigation result table (relative retention time)

Table 14 Centipede formula particle characteristic spectrum column temperature investigation result table (relative peak area)

4.2 Different flow Rate investigation

The effect of different flow rates (0.9 mL/min, 1.0mL/min and 1.1 mL/min) on the chromatograms was compared. A test solution was prepared as in example 2"4.4" by grinding the centipede formula particles to about 0.2 g. Other chromatographic conditions except for the flow rate were the same as those of the instrument of example 2 and the chromatographic conditions of example 2"1", the peak corresponding to the hypoxanthine reference peak, i.e., peak 4, was the S1 peak, and the relative retention times of peak 2, peak 3, peak 5 and S1 peak were calculated; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; and calculating the relative peak areas of the peaks 1-3, 5-9 and S1, and calculating the RSD value. The results are shown in Table 15, table 16 and FIG. 11. The result shows that the relative retention time RSD range of the peak 2, the peak 3, the peak 5 and the S1 peak is 0.23% -0.64%; the relative retention time RSD value of the peak 7, the peak 9 and the S2 peak is 0.10% -0.62%; the relative peak area RSD values of the peaks 1-3, 5-9 and S1 are 0.84% -3.35%; the relative retention time and relative peak area RSD values were < 5%, indicating that the qualitative method was better for both relative retention time and relative peak area durability when the flow rate was within a micro-variation range of + -0.1 mL/min.

Table 15 flow Rate investigation results table (relative retention time) for Scolopendra formulation granules

Table 16 flow velocity investigation results table (relative peak area) of centipede formula particle characteristic spectrum

4.3 Different chromatographic column investigation

The effect of column 1, column 2, column 3 on the chromatograms was compared. A test solution was prepared as in example 2"4.4" by grinding the centipede formula particles to about 0.2 g. Referring to the apparatus of example 2 and the chromatographic condition measurement under the item of example 2"1", peak 4 corresponding to the hypoxanthine reference peak is S1 peak, and the relative retention times of peak 2, peak 3, peak 5 and S1 peak are calculated; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; and calculating the relative peak areas of the peaks 1-3, 5-9 and S1, and calculating the RAD value. The comparison results of the chromatographic columns 1 and 2 are shown in tables 17, 18 and 12; the comparison results of the chromatographic main bodies 1 and 3 are shown in tables 19, 20 and 13.

Table 17 Scolopendra formulation particle characterization chromatographic column inspection result 1 (relative retention time)

Table 18 Scolopendra formulation particle characterization chromatographic column inspection result 1 (relative peak area)

As can be seen from table 17, table 18, fig. 12, the same model and different lot number chromatographic column measurements: the relative retention time RAD of the peak 2, the peak 3, the peak 5 and the S1 peak ranges from 0.06% to 0.68%; the relative retention time RAD value of the peak 7, the peak 9 and the S2 peak is 0.05% -0.31%; the relative peak area RAD values of the peaks 1-3, 5-9 and S1 are 0.27% -8.11%; the relative retention time RAD is less than 5%, and the qualitative method has good durability for different batches of JADE-PAK ODS-AQ chromatographic columns.

Table 19 Scolopendra formulation particle characterization chromatographic column inspection results 2 (relative retention time)

Table 20 Scolopendra formulation particle characteristics spectrum chromatographic column inspection result 2 (relative peak area)

As can be seen from table 19, table 20, fig. 13, 2 different types of chromatographic column assays: the relative retention time RAD of the peak 2, the peak 3, the peak 5 and the S1 peak ranges from 0.12% to 0.82%; the relative retention time RAD value of the peak 7, the peak 9 and the S2 peak is 0.21% -0.80%; the relative peak area RAD values of the peaks 1-3, 5-9 and S1 are 1.82% -18.37%; the relative retention time RAD is less than 5%, and the qualitative method has good durability to the relative retention time of chromatographic columns of different types.

Example 4 qualitative detection of samples and formulation of qualitative identification criteria

3 Batches of centipede formula particles (572303001 a, 572303002a, 572303003 a) were taken and a test solution was prepared as in example 2 "4.4". Respectively sucking the sample solution and the centipede reference solution, determining according to the chromatographic conditions under the instrument of the example 2 and the chromatographic conditions under the condition of the example 2"1', wherein the peak corresponding to the hypoxanthine reference peak, namely the peak 4 is the S1 peak, and calculating the relative retention time of the peak 2, the peak 3, the peak 5 and the S1 peak; peak 8, which is the peak corresponding to the guanosine control reference peak, is the S2 peak, and the relative retention times of peak 7, peak 9 and S2 peak are calculated; the relative peak areas of the peaks 1 to 3,5 to 9 and S1 were calculated, the RSD values were calculated, and the measurement results were shown in Table 21, table 22 and FIG. 14. The HPLC characteristic patterns of three batches of mass-produced centipede formula particles are matched by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, a comparison pattern is generated according to an average method, and the centipede formula particle comparison characteristic pattern is established, as shown in figure 15. The result shows that the characteristic patterns of the three batches of mass-produced centipede formula particles have the same 9 characteristic peaks, and the characteristic pattern standard (namely, the qualitative identification standard of liquid chromatography qualitative detection) of the centipede formula particles is determined as follows:

The sample chromatogram should show 9 characteristic peaks, peak 1, peak 4, peak 6, and peak 8 corresponding to uracil, hypoxanthine, uridine, and guanosine chromatogram peak retention times. Peak 4, which corresponds to the hypoxanthine reference peak, is the S1 peak, and the relative retention times of peak 2, peak 3, peak 5 and S1 peak are calculated to be within ±10% of the prescribed value, which is: 0.66 (Peak 2), 0.90 (Peak 3), 1.24 (Peak 5). Peak 8, which corresponds to the peak of the guanosine control reference, is the S2 peak, and the relative retention time of peak 7, peak 9, and S2 peak is calculated to be within ±10% of the prescribed value, which is: 0.97 (peak 7), 1.29 (peak 9).

Table 21 batch Scolopendra formula granule characteristics map (relative retention time)

Table 22 batch centipede formula particle characteristic spectrum (relative peak area)

As can be seen by combining the chromatograms of fig. 1 to 15 and the corresponding relative retention time data analysis, the qualitative method has high analysis efficiency (9 characteristic peaks are obtained in 40 minutes of elution time) and high quality control capability (9 characteristic peaks can be simultaneously analyzed and identified); and the characteristic peak separation degree well meets the requirements of qualitative analysis, the peak shape is good, and the base line is stable.

EXAMPLE 5 quantitative determination of samples and methodological investigation of quantitative determination

The quantitative detection target of the sample was hypoxanthine, and the chromatographic conditions for quantitative measurement, preparation of the sample, and preparation of the hypoxanthine reference solution were the same as in example 2.

1. Pretreatment method for test article

1.1 Investigation of extraction modes

Taking centipede formula particles, grinding, taking about 0.2g (two groups), weighing, placing into a conical bottle with a plug, adding 25mL of 10% methanol, sealing, weighing, respectively observing two modes of ultrasonic treatment (power 400W and frequency 40 kHz) and heating reflux, extracting for 30 minutes, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution. The hypoxanthine content was determined according to the apparatus of example 2 and the chromatographic conditions under the heading of example 2"1". As shown in Table 23, the hypoxanthine content obtained by the two extraction methods was substantially identical. Because the ultrasonic extraction is convenient to operate, the ultrasonic extraction is selected as an extraction mode for measuring the content of centipede formula particles.

Table 23 examination of different extraction modes for measuring the content of Scolopendra formula particles

1.2 Extraction solvent investigation

Taking centipede formula particles, grinding, taking about 0.2g (three groups), weighing, placing into a conical flask with a plug, respectively adding 25mL of 10% methanol, 30% methanol and 50% methanol, weighing, performing ultrasonic treatment (power 400W and frequency 40 kHz) for 30 minutes, cooling, weighing again, respectively adding corresponding solvents to supplement weight loss, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution. The hypoxanthine content was determined according to the apparatus of example 2 and the chromatographic conditions under the heading of example 2"1". As shown in Table 24, there was no significant difference in the amounts of hypoxanthine extracted with three concentrations of methanol, and 30% methanol was selected as the extraction solvent for the measurement of the amount of centipede particles, in combination with the convenience of the test results and the test operations.

Table 24 examination of extraction solvent for determining content of Scolopendra formulation particles

1.3 Extraction time investigation

Taking centipede formula particles, grinding, taking about 0.2g (three groups), weighing, placing into a conical bottle with a plug, adding 25mL of 30% methanol, sealing, weighing, respectively carrying out ultrasonic treatment (power 400W and frequency 40 kHz) for 15 minutes, 30 minutes and 45 minutes, cooling, supplementing the lost weight with 30% methanol, shaking uniformly, filtering, and taking the subsequent filtrate as a sample solution. The hypoxanthine content was determined according to the apparatus of example 2 and the chromatographic conditions under the heading of example 2"1". As shown in table 25, the data of ultrasonic extraction for 15 minutes, 30 minutes, and 45 minutes were not significantly different, so ultrasonic treatment for 15 minutes was selected as the extraction time for content measurement.

Table 25 examination of different extraction times for determining the content of Scolopendra formulation particles

1.4 Determination of the preparation method of the sample solution

In summary, the preparation method of the finally determined sample solution comprises the following steps: taking centipede formula particles, grinding, taking about 0.2g, weighing, placing into a conical flask with a plug, adding 25mL of 30% methanol, sealing, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 15 minutes, cooling, weighing again, supplementing the lost weight with 30% methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

2. Investigation of specificity

The auxiliary material of the centipede formula granule is maltodextrin. The influence of maltodextrin on quantitative detection was examined in this experiment. The maltodextrin and centipede formula particles were taken to prepare a negative solution and a test solution according to the method of example 2 ' 4.4 ', and the negative solution and the test solution were injected into a liquid chromatograph together with a hypoxanthine reference solution, and were measured according to the apparatus of example 2 and the chromatographic conditions under the item of example 2"1 '. And simultaneously, continuously running the same elution time according to the end point gradient proportion, examining the chromatographic peak condition after the specified time, and recording a chromatogram. See fig. 16, 17.

The result shows that maltodextrin has no interference to the content measurement of hypoxanthine, the separation degree of the hypoxanthine chromatographic peak and the adjacent chromatographic peak in the chromatogram of the test sample is more than 1.5, and the chromatogram of the test sample has no other chromatographic peak after 60min, so that the method has specificity in measuring the hypoxanthine content in the centipede formula particles.

3. Peak purity investigation

10 Μl of the sample solution was aspirated, and the sample solution was injected into the high performance liquid chromatograph 1, and the sample solution was subjected to scanning detection at 230nm to 270nm by a diode array detector (PDA) under the chromatographic conditions of example 2"1" to calculate the peak purity. The results are shown in FIG. 18. The purity angle (0.260) of the hypoxanthine peak in the chromatograph of the sample is smaller than the purity threshold (0.268), and is smaller than 1, and the purity of the chromatographic peak is qualified.

4. Linear relationship investigation

And (3) measuring 101.7 mug/mL of hypoxanthine reference substance stock solution, carrying out fractional dilution to reference substance solutions with different concentrations, sequentially injecting 10 mug of the reference substance solutions according to chromatographic conditions under the item of example 2"1", recording chromatographic peak areas, taking the peak areas as an ordinate (y), taking the reference substance concentrations as an abscissa (x), and drawing a standard curve. The regression equation is calculated as: y=41991x+6162.1, and the correlation coefficient r=0.9999. The linear relation between the concentration of the reference substance and the peak area is good within the range of 1.02-50.85 mug/mL.

5. Precision investigation

5.1 Precision of sample injection

The RSD value was calculated from retention time and hypoxanthine peak area by taking a hypoxanthine control solution and repeating the sample injection 6 times under the chromatographic conditions as described in example 2"1". As shown in Table 26, the retention time of hypoxanthine was 0.07% and the peak area was 0.33% with good instrument precision.

Table 26 examination result of precision of centipede formula particle content determination

5.2 Repeatability investigation

Taking centipede formula particles, grinding, taking about 0.2g, weighing, and preparing a test solution by using 6 parts in parallel according to the chromatographic condition of example 2 ' 4.4 ', and measuring the hypoxanthine content in the test solution according to the chromatographic condition of example 2"1 '. As shown in Table 27, the average hypoxanthine content was 1.488mg/g, RSD <2%, and the reproducibility of the analysis method was good.

Table 27 repetition of the measurement of the pellet content of the centipede formulation

5.3 Intermediate Density investigation

Different measurement times, different high performance liquid chromatographs and different experimenters are selected, centipede formula particles are taken, grinding is carried out, about 0.2g (6 parts total) is taken, a test solution is prepared according to the method of example 2 ' 4.4 ', the hypoxanthine content in the test solution is measured according to the chromatographic conditions under the method of example 2"1 ', and the comparison is carried out with the results under the repeated method. As shown in Table 28, the analytical methods were better in intermediate precision with different analytical personnel operating on different dates and with different chromatographs, with hypoxanthine content RSD < 2%.

Table 28 intermediate precision results of centipede formula particle content determination

Repeatability: personnel 1, instrument: high performance liquid chromatograph 1, numbered: JS-01-007; experimental date: 2022/05/06.

Intermediate precision: personnel 2, instrument: high performance liquid chromatograph 2, numbered: JS-01-012; experimental date: 2022/05/09.

6. Accuracy test

10.23Mg of hypoxanthine control was weighed, placed in a 100mL volumetric flask, dissolved with 10% methanol and diluted to the scale mark, to make a solution containing 101.70 μg per mL. And weighing 15-250 mL of the solution into a measuring flask, adding 30% methanol to dilute the solution to a scale, and preparing a control solution containing 6.101 mug of each mL of the solution. Taking centipede formula particles, grinding, taking about 0.1g, weighing 6 parts, respectively adding 25mL of the accuracy control solution, weighing, performing ultrasonic treatment (power 400W, frequency 40 kHz) for 15 minutes, cooling, weighing again, supplementing the weight loss with 30% methanol, shaking uniformly, filtering, and taking subsequent filtrate. The hypoxanthine content in the solution was measured under chromatographic conditions as in example 2"1", and the recovery rate of the sample was calculated. As shown in Table 29, the hypoxanthine sample recovery rate is 100.59% -102.22%, the average sample recovery rate is 101.54%, the RSD is 0.55%, and the accuracy of the method is high.

Table 29 results of sample recovery from Centipede formula particle content determination

7. Stability investigation

Taking centipede formula particles, grinding, taking about 0.2g, weighing, preparing a test solution according to the example 2 of 4.4, respectively carrying out sample injection and measurement on the peak areas of hypoxanthine according to chromatographic conditions under the items of example 2"1 at 0, 10, 30, 40 and 52 hours, and calculating the peak area RSD. As shown in Table 30, the RSD of the hypoxanthine peak area was less than 2%, and the test solution was stable and measurable over 52 hours.

Table 30 stability test results of test sample for determining the content of Scolopendra formula particles

8. Durability inspection

8.1 Different column temperature investigation

Taking centipede formula particles, grinding, taking about 0.2g, weighing, preparing a test solution according to the method of example 2 ' 4.4 ', measuring the other components according to chromatographic conditions under the condition of example 2"1 ' except that the column temperature is 28 ℃ and 30 ℃ respectively and 32 ℃, and comparing the influence of different column temperatures on the measurement of hypoxanthine content. As shown in Table 31, the hypoxanthine content RSD was 4.00% at three different column temperatures, and the column temperature was set to 30℃in accordance with the requirement.

Table 31 examination results of different column temperature durability tests of centipede formula particle content measurement

8.2 Different flow Rate investigation

The effect of different flow rates of 0.9mL/min, 1.0mL/min and 1.1mL/min on the hypoxanthine content determination was compared. The sample solutions in the "durability test" of this example (different column temperatures) were taken and the hypoxanthine content was measured under the chromatographic conditions under the item 2"1" of example except for the flow rate. As shown in Table 32, the hypoxanthine content RSD was 4.08% at three different flow rates, and the flow rate setting of the analysis method was 1.0mL/min as required.

Table 32 examination results of the determination of the content of Scolopendra formulation particles and the durability at different flow rates

8.3 Different chromatographic column investigation

Comparing the influence of the chromatographic column 1, the chromatographic column 2 and the chromatographic column 3 on the content measurement of centipede formula particles. Taking centipede formula particles, grinding, taking about 0.2g, weighing, preparing a test solution according to the method of example 2 ' 4.4 ', and measuring the hypoxanthine content according to the chromatographic conditions under the item of example 2"1 '. As shown in Table 33, the three columns have a hypoxanthine content RSD of 1.86% and RSD value < 2%, and the method has good durability to the three columns.

Table 33 examination results of different chromatographic columns for measuring content of centipede formula particles

9. Knot (S)

Quantitative measurement methodology investigation of hypoxanthine in centipede formula particles shows that the color spectrum condition is blank without interference and has good specificity; the theoretical plate number is more than 10000, and the hypoxanthine has good linearity in the concentration range of 1.02-50.85 mug/mL; the sample injection precision, repeatability and intermediate precision are good; the test solution is stable at room temperature for 52 hours; the recovery rate is 100.59-102.22%, and the accuracy is high; the column temperature is 30 ℃, the flow rate is 1.0mL/min, and the durability is good. The durability to different chromatographic columns is good. The method can be accurately used for quantitative determination of hypoxanthine in centipede formula particles.

Claims (10)

1. A liquid chromatography qualitative detection method of centipede formula particles is characterized by comprising the following steps:

preparing a test solution: taking centipede formula particles, carrying out ultrasonic or heating reflux extraction by adopting a methanol aqueous solution with the concentration of 10% -50%, filtering, and taking a subsequent filtrate as a sample solution;

preparing a reference substance solution: dissolving hypoxanthine reference substance, uracil reference substance, uridine reference substance and guanosine reference substance in methanol water solution to obtain reference substance solution;

And (3) sample injection detection: taking sample solutions of the sample and the reference substance respectively; the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler, and the parameters of the chromatographic column are as follows: the column length is 250mm, the inner diameter is 4.6mm, and the grain diameter is 5 μm; methanol is used as a mobile phase A, and 0.1% glacial acetic acid solution is used as a mobile phase B, and gradient elution is carried out; the flow rate is 0.9-1.1 mL/min, the column temperature is 28-32 ℃, and the detection wavelength is 248nm; the elution procedure included the following: 0-15 minutes: the volume percentage of the mobile phase A is 0%;15 minutes to 30 minutes: the volume percent ratio of the mobile phase A is increased from 0% to 10%; 30-45 minutes: the volume percentage of the mobile phase A is increased from 10% to 40%.

2. The method for qualitatively detecting centipede particles according to claim 1, wherein the extraction time is 15-45 minutes in the step of preparing the sample solution.

3. The method for qualitatively detecting centipede particles according to claim 1, wherein in the step of preparing the sample solution, the extraction method is ultrasonic extraction; the ultrasonic power is 400W and the frequency is 40kHz.

4. The method for qualitatively detecting a centipede granule according to claim 1, wherein the concentration of the aqueous methanol solution in the step of preparing the reference solution is 10%.

5. The method for qualitatively detecting centipede particles according to claim 4, wherein the concentration of hypoxanthine, uracil, uridine and guanosine in the reference solution is 10 μg,5 μg, 8 μg and 8 μg per mL in the reference solution.

6. The liquid chromatography quantitative detection method of the centipede formula particles is characterized by comprising the following steps of:

preparing a test solution: taking centipede formula particles, carrying out ultrasonic or heating reflux extraction by adopting a methanol aqueous solution with the concentration of 10% -50%, filtering, and taking a subsequent filtrate as a sample solution;

Preparing a reference substance solution: dissolving hypoxanthine reference substance in methanol water solution to obtain hypoxanthine reference substance solution;

And (3) sample injection detection: taking sample solutions of the sample and the hypoxanthine reference substance, respectively injecting samples, and measuring the hypoxanthine content in the sample solutions; the chromatographic column takes octadecylsilane chemically bonded silica gel as a filler, and the parameters of the chromatographic column are as follows: the column length is 250mm, the inner diameter is 4.6mm, and the grain diameter is 5 μm; methanol is used as a mobile phase A, and 0.1% glacial acetic acid solution is used as a mobile phase B, and gradient elution is carried out; the flow rate is 1.0 mL/min, the column temperature is 30 ℃, and the detection wavelength is 248nm; the elution procedure included the following: 0-15 minutes: the volume percentage of the mobile phase A is 0%;15 minutes to 30 minutes: the volume percent ratio of the mobile phase A is increased from 0% to 10%; 30-45 minutes: the volume percentage of the mobile phase A is increased from 10% to 40%.

7. The method for quantitatively detecting centipede formula particles by liquid chromatography according to claim 6, wherein the extraction time is 15-45 minutes in the preparation step of the sample solution.

8. The method for quantitatively detecting centipede formula particles by liquid chromatography according to claim 6, wherein in the step of preparing the sample solution, the extraction method is ultrasonic extraction; the ultrasonic power is 400W and the frequency is 40kHz.

9. The method for quantitatively detecting centipede formula particles by liquid chromatography according to claim 6, wherein the concentration of the aqueous solution of methanol in the step of preparing the reference solution is 10%.

10. The method for quantitatively detecting centipede formula particles by liquid chromatography according to claim 9, wherein the concentration of hypoxanthine in the hypoxanthine reference solution is 10 μg/mL.