Construction of HPLC characteristic chromatogram of Jiaoliushenqu medicinal materials, pieces, formula granules and standard decoction and determination of adenine content

Abstract

The application provides a construction method of HPLC characteristic spectrum of Jiaoliushenqu medicinal materials, decoction pieces and standard decoction and a determination method of adenine content, which comprises the following steps: extracting a test sample raw material by using 30% methanol to obtain a to-be-tested solution; determining the to-be-tested solution by using HPLC to obtain the HPLC characteristic spectrum of Jiaoliushenqu medicinal materials, decoction pieces and standard decoction; and preparing reference solution by respectively dissolving uracil, adenine and 5-hydroxymethylfurfural control samples by using a solvent to obtain the control sample reference solution; the HPLC conditions are that a C 18 column is used as the chromatographic column, the mobile phase A is acetonitrile solution, the mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is adopted. The application adopts the high performance liquid chromatography method, selects acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution, takes uracil, adenine and 5-hydroxymethylfurfural as the reference objects, establishes the HPLC characteristic spectrum method of Jiaoliushenqu medicinal materials, decoction pieces and standard decoction, and provides more scientific technical means for controlling the medicinal quality of Jiaoliushenqu medicinal materials, decoction pieces and standard decoction.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical and formulation analysis and detection technology, and in particular to a method for constructing HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, formulation granules, and standard decoctions, as well as a method for determining adenine content. Background Technology

[0002] Shenqu (a type of medicinal fermented wheat) has the effects of strengthening the spleen and stomach, promoting qi circulation and digestion, and relieving exterior symptoms. It is often used for indigestion, abdominal distension, poor appetite and diarrhea, poor appetite, or food stagnation combined with external pathogenic factors. After stir-frying, its spleen-strengthening and stomach-nourishing functions are enhanced, while its dispersing effect is weakened.

[0003] Currently, there are no relevant standards for the determination of the content of Jiao Liu Shen Qu (a type of fermented wheat bran) in medicinal slices. The relevant literature mainly focuses on the study of alkaloids and aromatic components, while there is a lack of literature on the qualitative and quantitative research of Jiao Liu Shen Qu formula granules and related preparations.

[0004] The present invention aims to construct a high-performance liquid chromatography method for the determination of the content and characteristic chromatogram of charred shenqu, which comprehensively reflects the quality level of charred shenqu and its related preparations, and provides guidance for the quality control of charred shenqu slices and its related preparations. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, and standard decoctions, and for determining adenine content. The specific chromatogram identification results provided by the present invention are accurate, and the determination of adenine content is accurate and reliable.

[0006] A method for constructing HPLC characteristic chromatograms of charred Shenqu (a type of medicinal herb), processed medicinal slices, and standard decoctions, comprising:

[0007] The raw sample was extracted with 30% methanol to obtain the test solution; the test solution was analyzed by HPLC to obtain the HPLC characteristic chromatograms of the charred Shenqu medicinal material, decoction pieces, and standard decoction.

[0008] Preparation of reference solutions: Uracil, adenine, and 5-hydroxymethylfurfural reference standards were dissolved in solvents to obtain reference solutions;

[0009] The HPLC conditions were as follows: a C18 column; mobile phase A was acetonitrile solution, mobile phase B was 0.1% phosphoric acid aqueous solution, and gradient elution was used.

[0010] The gradient elution specifically refers to:

[0011] 0–10 min, Phase A: 1% → 5%, Phase B: 99% → 95%;

[0012] 10–18 min, Phase A: 5% → 8%, Phase B: 95% → 92%;

[0013] 18–25 min, Phase A: 8% → 14%, Phase B: 92% → 86%;

[0014] 25–30 min, Phase A: 14%, Phase B: 86%.

[0015] The raw materials for the test sample described in this invention are one or more of the following: charred Shenqu medicinal materials, processed medicinal slices, and standard decoctions.

[0016] This invention provides a method for constructing HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, and standard decoctions. First, the raw materials for testing are pretreated. The pretreatment method includes ultrasonic treatment with 30% methanol, cooling, shaking, filtering, and taking the filtrate as the test solution.

[0017] The ultrasonic extraction device of the present invention is set with an electrical power of 600W and an ultrasonic vibration frequency of 40kHz; the duration of ultrasonic extraction is 30 minutes.

[0018] The present invention achieves complete extraction under the above-mentioned ultrasonic parameters. The chromatogram shows good peak shape and resolution.

[0019] The ratio of the mass (g) of the test sample raw material to the volume (mL) of methanol in this invention is 1g:25mL.

[0020] When using the extraction solvent and specific solvent addition amounts, the present invention achieves good peak shape and resolution, and moderate peak size for each chromatographic peak.

[0021] All the raw materials mentioned above can be subjected to quality control using the methods of this invention.

[0022] The present invention also includes the preparation of reference solutions: uracil, adenine, and 5-hydroxymethylfurfural reference standards are dissolved in a solvent to obtain reference solutions;

[0023] The reference standard solution was injected into a high-performance liquid chromatograph for determination, and the chromatogram of the reference standard was obtained. Based on the characteristic parameters of the chromatogram of the reference standard, the corresponding chromatograms in the HPLC characteristic chromatograms of the scorched ginseng medicinal material, decoction pieces, standard decoction medicinal material test sample, standard decoction test sample, and formula granule test sample were compared and identified.

[0024] The preferred concentrations of the reference solution in this invention are: uracil 20 μg / mL, adenine 20 μg / mL, and 5-hydroxymethylfurfural 20 μg / mL.

[0025] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of the charred Shenqu medicinal material, decoction pieces, and standard decoction.

[0026] The chromatographic conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is a C18 column; the chromatographic column is a C18 column. 18 The specifications are 150×2.1mm 2μm; this invention does not limit the specific chromatographic column model, any C18 chromatographic column that meets the above specifications is acceptable.

[0027] In some embodiments, the chromatographic columns were Shimadzu Shim-pack GIS C18, 2.1 mm × 150 mm, 2 μm; Shimadzu Shim-pack GIST C18, 2.1 mm × 150 mm, 2 μm; or Shimadzu Inertsil ODS-3, 2.1 mm × 150 mm, 2 μm.

[0028] The preferred column temperature for this invention is 25°C. At the above-mentioned column temperature, the chromatographic peaks are symmetrical, the separation is good, and the elution is complete.

[0029] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution.

[0030] Specifically, the gradient elution is as follows:

[0031] 0–10 min, Phase A: 1% → 5%, Phase B: 99% → 95%;

[0032] 10–18 min, Phase A: 5% → 8%, Phase B: 95% → 92%;

[0033] 18–25 min, Phase A: 8% → 14%, Phase B: 92% → 86%;

[0034] 25–30 min, Phase A: 14%, Phase B: 86%.

[0035] The theoretical plate number, calculated based on adenine, should be no less than 3000.

[0036] The invention creatively discovers that, under the above-mentioned elution gradient, the baseline separation is good, the separation between each peak is good, the distribution is uniform, and the baseline is stable.

[0037] The flow rate of the mobile phase described in this invention is 1.0 mL / min.

[0038] The present invention found that the chromatographic peaks were well separated, with moderate resolution and complete elution at the above-mentioned flow rates, which is the optimal solution.

[0039] The injection volume was 2 μL.

[0040] The detection wavelength is 260 nm. The inventors have discovered that at 260 nm, the chromatographic peak information content is greater, the chromatographic baseline is more stable, and the peak areas are larger.

[0041] This invention uses a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine to evaluate the similarity of HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, and standard decoctions. A standard HPLC characteristic chromatogram consisting of 12 characteristic peaks was constructed, and the chemical composition information corresponding to each characteristic peak is as follows: peak 1 is uracil, peak 4 is adenine, and peak 5 is 5-hydroxymethylfurfural.

[0042] The peak corresponding to the adenine reference standard peak is the S peak. Calculate the relative retention time of each of the other characteristic peaks and the S peak. The relative retention time should be within ±10% of the specified value: the specified values ​​are: 0.64 (peak 2), 0.77 (peak 3), and 1.95 (peak 6).

[0043] Quality judgment criteria: Take the raw materials, decoction pieces, standard decoctions, and their processed products of Jiao Liu Shen Qu (a type of medicinal fermented wheat), extracts and their preparations, and operate according to the same method as above to obtain the above characteristic chromatograms. Analyze the characteristic chromatograms of the raw materials, decoction pieces, standard decoctions, and their processed products of Jiao Liu Shen Qu and their preparations, as well as the sample characteristic chromatograms, using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" issued by the National Pharmacopoeia Commission. The similarity is greater than 0.90.

[0044] This invention provides a method for determining the content of charred Shenqu (a type of medicinal fermented wheat), its processed forms, and standard decoctions, comprising the following steps:

[0045] The raw sample was extracted with 30% methanol to obtain the test solution; the content of the charred Shenqu medicinal material, decoction pieces and standard decoction was determined by HPLC.

[0046] Preparation of reference solutions: Adenine, uracil, and 5-hydroxymethylfurfural reference standards were dissolved in solvents to obtain reference solutions;

[0047] The HPLC conditions were as follows: a C18 column; mobile phase A was acetonitrile solution, mobile phase B was 0.1% phosphoric acid aqueous solution, and gradient elution was used.

[0048] The gradient elution specifically refers to:

[0049] 0–10 min, Phase A: 1% → 5%, Phase B: 99% → 95%;

[0050] 10–18 min, Phase A: 5% → 8%, Phase B: 95% → 92%;

[0051] 18–25 min, Phase A: 8% → 14%, Phase B: 92% → 86%;

[0052] 25–30 min, Phase A: 14%, Phase B: 86%.

[0053] First, the raw materials for the test sample are pretreated. The pretreatment method includes ultrasonic treatment with 30% methanol, cooling, shaking, filtering, and taking the filtrate as the test sample solution.

[0054] The ultrasonic extraction device of the present invention is set with an electrical power of 600W and an ultrasonic vibration frequency of 40kHz; the duration of ultrasonic extraction is 30 minutes.

[0055] The present invention achieves complete extraction under the above-mentioned ultrasonic parameters. The chromatogram shows good peak shape and resolution.

[0056] The ratio of the mass (g) of the test sample raw material to the volume (mL) of methanol in this invention is 1g:25mL.

[0057] When using the extraction solvent and specific solvent addition amounts, the present invention achieves good peak shape and resolution, and moderate peak size for each chromatographic peak.

[0058] All the raw materials mentioned above can be subjected to quality control using the methods of this invention.

[0059] The present invention also includes the preparation of a reference solution: adenine reference standard is dissolved in a solvent to obtain a reference solution;

[0060] The reference standard solution was injected into a high-performance liquid chromatograph for determination, and the chromatogram of the reference standard was obtained. Based on the characteristic parameters of the chromatogram of the reference standard, the corresponding chromatograms in the HPLC characteristic chromatograms of the scorched ginseng medicinal material, decoction pieces, standard decoction medicinal material test sample, standard decoction test sample, and formula granule test sample were compared and identified.

[0061] When the adenine concentration range of this invention is 1.4981976~37.45494μg / ml, the linear relationship is y = 53.58x+1.5138, R²=1.

[0062] When the concentration of uracil was in the range of 2.163664~43.27328 μg / ml, the linear relationship was y = 20.483x +9.2842, R²=0.9999;

[0063] When the concentration of 5-hydroxymethylfurfural was in the range of 4.485012~89.70024 μg / ml, the linear relationship was y = 12.029x + 10.304, R² = 0.9999.

[0064] The test solution was analyzed by high performance liquid chromatography to obtain the HPLC characteristic chromatograms of the charred Shenqu medicinal material, decoction pieces, and standard decoction.

[0065] The chromatographic conditions for the high-performance liquid chromatography (HPLC) are as follows: the chromatographic column is a C18 column; the chromatographic column is a C18 column. 18The specifications are 150×2.1mm 2μm; this invention does not limit the specific chromatographic column model, any C18 chromatographic column that meets the above specifications is acceptable.

[0066] In some embodiments, the chromatographic columns were Shimadzu Shim-pack GIS C18, 2.1 mm × 150 mm, 2 μm; Shimadzu Shim-pack GIST C18, 2.1 mm × 150 mm, 2 μm; or Shimadzu Inertsil ODS-3, 2.1 mm × 150 mm, 2 μm.

[0067] The preferred column temperature for this invention is 25°C. At the above-mentioned column temperature, the chromatographic peaks are symmetrical, the separation is good, and the elution is complete.

[0068] In this invention, mobile phase A is an acetonitrile solution, and mobile phase B is a 0.1% phosphoric acid aqueous solution, with gradient elution.

[0069] Specifically, the gradient elution is as follows:

[0070] 0–10 min, Phase A: 1% → 5%, Phase B: 99% → 95%;

[0071] 10–18 min, Phase A: 5% → 8%, Phase B: 95% → 92%;

[0072] 18–25 min, Phase A: 8% → 14%, Phase B: 92% → 86%;

[0073] 25–30 min, Phase A: 14%, Phase B: 86%.

[0074] The theoretical plate number, calculated based on adenine, should be no less than 3000.

[0075] The invention creatively discovers that, under the above-mentioned elution gradient, the baseline separation is good, the separation between each peak is good, the distribution is uniform, and the baseline is stable.

[0076] The flow rate of the mobile phase described in this invention is 1.0 mL / min.

[0077] The present invention found that the chromatographic peaks were well separated, with moderate resolution and complete elution at the above-mentioned flow rates, which is the optimal solution.

[0078] The injection volume was 2 μL.

[0079] The detection wavelength is 260 nm. The inventors have discovered that at 260 nm, the chromatographic peak information content is greater, the chromatographic baseline is more stable, and the peak areas are larger.

[0080] Compared with the prior art, the present invention provides a method for constructing HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, and standard decoctions, as well as a method for determining adenine content. The method for constructing HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, and standard decoctions includes: extracting the test sample raw material with 30% methanol to obtain a test solution; determining the test solution using HPLC to obtain HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, and standard decoctions; and preparing a reference solution: dissolving uracil, adenine, and 5-hydroxymethylfurfural reference standards in a solvent to obtain a reference solution; the HPLC conditions are: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is used. This invention employs high-performance liquid chromatography (HPLC) with acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution. Using uracil, adenine, and 5-hydroxymethylfurfural as references, a method for establishing HPLC characteristic chromatograms of charred shenqu (a type of medicinal preparation), its processed slices, and standard decoctions was established. This provides more scientific technical means for controlling the medicinal quality of charred shenqu (a type of medicinal preparation), its processed slices, and standard decoctions.

[0081] This invention also provides a method for determining the content of charred Shenqu (a type of medicinal herb), processed medicinal pieces, and standard decoction, comprising the following steps: extracting the test sample raw material with 30% methanol to obtain the test solution; determining the content of charred Shenqu, processed medicinal pieces, and standard decoction by HPLC; and preparing the reference solution: dissolving adenine reference standard in a solvent to obtain the reference solution; the HPLC conditions are: a C18 column; mobile phase A is acetonitrile solution, mobile phase B is 0.1% phosphoric acid aqueous solution, and gradient elution is used. This invention uses high-performance liquid chromatography, selecting acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution, resulting in accurate and reliable adenine determination. Attached Figure Description

[0082] Figure 1 Chromatograms at different wavelengths;

[0083] Figure 2 Investigation at different column temperatures;

[0084] Figure 3 Investigation at different flow velocities;

[0085] Figure 4 Delayed investigation;

[0086] Figure 5 Extraction solvent investigation;

[0087] Figure 6 Examining the extraction method;

[0088] Figure 7 Extraction time consideration;

[0089] Figure 8 Sampling size consideration;

[0090] Figure 9 Chromatographic peak identification;

[0091] Figure 10 Exploration using different instruments;

[0092] Figure 11 Investigations using different chromatographic columns;

[0093] Figure 12 Formulation particle validation;

[0094] Figure 13 adenine ultraviolet absorption spectrum;

[0095] Figure 14 Column temperature investigation results

[0096] Figure 15 Flow velocity study results;

[0097] Figure 16 Specificity graph;

[0098] Figure 17-1 Adenine standard curve;

[0099] Figure 17-2 Uracil standard curve;

[0100] Figure 17-3 This is a standard curve for 5-hydroxymethylfurfural;

[0101] Figure 18 Comparative characteristic spectrum of Jiao Liu Shen Qu formula granules;

[0102] Figure 19 Comparative characteristic atlas of charred Shenqu (medicinal fermentation) material;

[0103] Figure 20 Comparative characteristic spectrum of standard decoction of Jiao Liu Shen Qu;

[0104] Figure 21 This is a diagram showing the results of the comparative method of the present invention. Detailed Implementation

[0105] This invention provides a method for constructing HPLC characteristic chromatograms of charred Shenqu (a type of medicinal herb), processed medicinal slices, and standard decoctions. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0106] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing HPLC characteristic chromatograms of charred Shenqu medicinal materials, decoction pieces, and standard decoctions provided by the present invention.

[0107] High performance liquid chromatographs: Waters UPLC H-class, Agilent 1290, Thermo Fisher Vanquish Flex;

[0108] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Ltd.);

[0109] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0110] Ultrasonic cleaner: KQ5200DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0111] Chromatographic columns: Shimadzu Shim-pack GIS C18, 2.1mm×150mm, 2μm; Shimadzu Shim-pack GIST C18, 2.1mm×150mm, 2μm; Shimadzu Inertsil ODS-3, 2.1mm×150mm, 2μm.

[0112] Uracil reference standard (China National Institutes for Food and Drug Control, batch number: 100469-201302, purity: 99.6%).

[0113] Adenine reference standard (China National Institutes for Food and Drug Control, batch number: 110886-202404, purity: 100.0%).

[0114] 5-Hydroxymethylfurfural (China National Institutes for Food and Drug Control, batch number: 111626-202316, purity: 99.4%)

[0115] Artemisia annua reference material (China National Institutes for Food and Drug Control, batch number: 121016-202007).

[0116] Red adzuki bean reference material (China National Institutes for Food and Drug Control, batch number: 121725-202103);

[0117] Spicy Liao reference medicinal material (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 310096-202208);

[0118] Bitter almond reference material (China National Institutes for Food and Drug Control, batch number: 121554-201204).

[0119] Xanthium sibiricum reference material (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYC000201);

[0120] Jiao Liu Shen Qu (a type of traditional Chinese medicine) formula granules: JLSQ-KL-01, JLSQ-KL-02, JLSQ-KL-03

[0121] Liushenqu medicinal materials: LSQ-YC-01, LSQ- YC -02, LSQ- YC -03, LSQ- YC -04, LSQ- YC -05, LSQ- YC -06, LSQ- YC -07, LSQ- YC -08, LSQ- YC -09, LSQ- YC -10, LSQ- YC -11, LSQ-YC -12, LSQ- YC -13, LSQ- YC -14, LSQ- YC -15, LSQ- YC -16, LSQ- YC -17, LSQ- YC -18, LSQ- YC -19, LSQ- YC -20, LSQ- YC -21, LSQ- YC -22, LSQ- YC -23;

[0122] Jiao Liu Shen Qu decoction pieces: JLSQ-YP-01, JLSQ-YP-02, JLSQ-YP-03, JLSQ-YP-04, JLSQ-YP-05, JLS Q-YP-06, JLSQ-YP-07, JLSQ-YP-08, JLSQ-YP-09, JLSQ-YP-10, JLSQ-YP-11, JL SQ-YP-12, JLSQ-YP-13, JLSQ-YP-14, JLSQ-YP-15, JLSQ-YP-16, JLSQ-YP-17, J LSQ-YP-18, JLSQ-YP-19, JLSQ-YP-20, JLSQ-YP-21, JLSQ-YP-22, JLSQ-YP-23;

[0123] Standard decoction of Jiao Liu Shen Qu: JLSQ-BT-01, JLSQ-BT-02, JLSQ-BT-03, JLSQ-BT-04, JLSQ-BT-05, JLSQ-BT-06, JLSQ-BT-07, JLSQ-BT-08, JLSQ-BT-09, JLSQ-BT-10, JLSQ-BT-11, JLSQ-BT-12, JLSQ-BT-13, JLSQ-BT-14, JLSQ-BT-15, JLSQ-BT-16, JLSQ-BT-17, JLSQ-BT-18, JLSQ-BT-19, JLSQ-BT-20, JLSQ-BT-21, JLSQ-BT-22, JLSQ-BT-23.

[0124] Example 1

[0125] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 2 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.20 mL / min; column temperature 25 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the adenine peak, should be no less than 3000.

[0126]

[0127] Preparation of reference solution: Take appropriate amounts of uracil, adenine, and 5-hydroxymethylfurfural reference standards, accurately weigh them, and add 30% methanol to prepare a mixed solution containing 20µg of each per ml, which is used as the reference solution.

[0128] Preparation of the test solution: Take an appropriate amount of Jiao Liu Shen Qu formula granules, about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 30% methanol, stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 30% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0129] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0130] The chromatogram of the test sample should show six characteristic peaks. Peaks 1, 4, and 5 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the adenine reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.64 (peak 2), 0.77 (peak 3), and 1.95 (peak 6).

[0131] 2.2.2 Determination of Detection Wavelength Point

[0132] Based on the above-planned experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, and 300 nm were extracted. Figure 1 As shown. Figure 1 Chromatograms at different wavelengths.

[0133] The results showed that the chromatographic peak information was greater and the chromatographic baseline was more stable at a detection wavelength of 260 nm, so the detection wavelength was determined to be 260 nm.

[0134] 2.2.3 Column Temperature Investigation

[0135] Based on the above-planned experimental conditions, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃. Figure 2 As shown. Figure 2 The study investigated different column temperatures. Results showed that at a column temperature of 25℃, the chromatographic peaks were generally symmetrical, with good overall peak shape and good peak separation. Therefore, a column temperature of 25℃ was initially selected for further research.

[0136] 2.2.4 Flow velocity investigation

[0137] Based on the above-established experimental conditions, the flow rates of 0.2 ml / min, 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min were investigated. Figure 3 As shown. Figure 3 Different flow rates were investigated; the results showed that when the flow rate was 0.20 ml / min, the overall peak shape of the chromatogram was better and the separation effect was better. Therefore, a flow rate of 0.20 ml / min was selected for further research.

[0138] 2.2.5 Delayed testing

[0139] Based on the above-planned experimental conditions, the chromatogram acquisition time was extended to 140 minutes. For example... Figure 4 As shown. Figure 4 Delayed examination

[0140] The results showed that the sample had virtually no chromatographic peaks after 30 minutes, so the sample detection time was set at 30 minutes.

[0141] 2.2.6 Examination of Extraction Methods

[0142] Take 1.0g of the granulated formula of Jiao Liu Shen Qu (JLSQ-KL-01) and grind it into a fine powder. Place it in an Erlenmeyer flask and add 25ml of 30% methanol. Examine the extraction methods for reflux and ultrasonic treatment (600W power, 40kHz frequency) for 30 minutes. After cooling, collect the filtrate to obtain the product. Figure 5 As shown. Figure 5 Extraction solvent investigation: The results showed that there was little difference in the effectiveness of reflux and ultrasonic extraction of the test sample, and the ultrasonic method was fast and simple. Therefore, ultrasonic extraction was selected as the extraction method for the test sample.

[0143] 2.2.7 Investigation of Extraction Solvents

[0144] Take 1.0g of the granulated formula of Jiao Liu Shen Qu (JLSQ-KL-01) and grind it into a fine powder. Place it in an Erlenmeyer flask and test it with 25ml each of methanol, 10% methanol, 30% methanol, 50% methanol, 80% methanol, water, and 50% ethanol as the extraction solvents. Seal the flask tightly, weigh it, and sonicate it (600W, 40kHz) for 30 minutes. After cooling, collect the filtrate to obtain the product. Figure 6 As shown. Figure 6 Extraction method investigation; the results showed that when the extraction solvent was 30% methanol, the overall peak shape of the chromatographic peak was better and the separation effect was better. Therefore, the extraction solvent was tentatively set as 30% methanol.

[0145] 2.2.8 Examination of extraction time

[0146] Grind this product (JLSQ-KL-01) into a fine powder. Take 1.0g and place it in an Erlenmeyer flask. Add 25ml of 30% methanol and sonicate (600W power, 40kHz frequency). Examine the extraction time at 15 minutes, 30 minutes, and 45 minutes. Cool and collect the filtrate. This is the product. Figure 7 As shown. Figure 7 Extraction time was investigated; the results showed that extraction times of 15 minutes, 30 minutes, and 45 minutes resulted in better peak shapes and resolution in the chromatograms. An extraction time of 30 minutes was tentatively selected for further investigation.

[0147] 2.2.9 Investigation of Solvent Addition Amount

[0148] Grind this product (JLSQ-KL-01) into a fine powder. Take 1.0g and place it in a stoppered conical flask. Add 10ml, 25ml, and 50ml of 30% methanol respectively for further analysis. Sonicate the mixture (600W power, 40kHz frequency) for 15 minutes, cool, and collect the filtrate. This is the final product. Figure 8 As shown. Figure 8 Sampling volume analysis. The results showed that the characteristic peaks were better when the solvent volume was 25 ml, therefore, 25 ml of solvent was selected.

[0149] 2.2.10 Determine the method for preparing the test sample

[0150] Take an appropriate amount of Jiao Liu Shen Qu formula granules, grind them into a fine powder, take about 1.0g, place it in an Erlenmeyer flask, add 25ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, and collect the filtrate to obtain the final product.

[0151] 2.2.11 Methodological Examination

[0152] 2.2.11.1 Chromatographic Peak Identification

[0153] Preparation of the test solution: Prepare the test solution of Jiao Liu Shen Qu formula granules according to the experimental conditions proposed above.

[0154] Preparation of reference solutions: Take 1g of Artemisia annua, adzuki bean, Lysimachia christinae, bitter almond, and Xanthium sibiricum reference materials, add 25ml of 30% methanol to each, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, filter, and take the filtrate as the reference solution for the reference materials.

[0155] Take appropriate amounts of adenine reference standard, uracil reference standard, and 5-hydroxymethylfurfural reference standard, accurately weigh them, and add 30% methanol to prepare a solution containing 20 μg of each per ml, which will be used as the reference solution.

[0156] Preparation of negative control solution: Prepare a negative control solution of the pyrolysis-deficient Liu Shen Qu formula granules according to the experimental conditions outlined above.

[0157] The characteristic peaks of the granule composition of Jiao Liu Shen Qu were located. For example... Figure 9 As shown. Figure 9 Chromatographic peak identification. The characteristic peaks in the Jiao Liu Shen Qu formula granules are derived from other single herbs. Peak 1 mainly comes from Xanthium sibiricum and Artemisia annua. Peaks 2 and 4 are present in bitter almond, Xanthium sibiricum, Artemisia annua, red adzuki bean, and Polygonum hydropiper. Peak 3 mainly comes from Artemisia annua. Peak 5 is a new component produced after the Jiao Liu Shen Qu medicinal material is processed into Jiao Liu Shen Qu decoction pieces. Peak 6 mainly comes from red adzuki bean and bitter almond.

[0158] The results showed that peak 1 was uracil, peak 4 was adenine, and peak 5 was 5-hydroxymethylfurfural. In the following methodological investigation, six characteristic peaks in the sample were examined.

[0159] 2.2.11.2 Precision Test

[0160] Take the test solution of Jiao Liu Shen Qu formula granules (batch number: JLSQ-KL-01), and inject it 6 times consecutively according to the proposed experimental method, 2 μl each time. Calculate the retention time and peak area of ​​each characteristic peak. As shown in Table 1.

[0161] Table 1 Precision test - retention time

[0162]

[0163] The results show that the instrument has good precision.

[0164] 2.2.11.3 Repeatability Test

[0165] Six portions of Jiao Liu Shen Qu formula granules (batch number: JLSQ-KL-01) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 2 for details.

[0166] Table 2 Repeatability test - relative retention time ratio

[0167]

[0168] The results show that the method has good repeatability.

[0169] 2.2.11.4 Intermediate Precision Examination

[0170] 2.2.11.4.1 Investigation with different instruments

[0171] Based on the above-planned experimental conditions, precisely weigh the granules of Jiao Liu Shen Qu (batch number: JLSQ-KL-01) to prepare test solutions, and perform determinations on Agilent 1290, Thermo Fisher Scientific, and Shimadzu ultra-high performance liquid chromatographs, respectively. Figure 10 As shown in Table 3.

[0172] Table 3 Instrument durability test - relative retention time ratio

[0173]

[0174] Figure 10 Different instruments were used for the investigation; the results showed that when the test sample was detected using the above three instruments, the RSD of the relative retention time of each characteristic peak was less than 10%.

[0175] 2.2.11.4.2 Investigations by different personnel and at different times

[0176] Based on the experimental conditions outlined above, different personnel (A and B) precisely weighed the granules of Jiao Liu Shen Qu (batch number: JLSQ-KL-01) at different times (T1 and T2) to prepare test samples for determination. The results are shown in Table 4.

[0177] Table 4. Personnel and Time Assessment - Ratio of Relative Retention Time

[0178]

[0179] The results show that the method has good stability when different people measure the same sample at different times.

[0180] 2.2.11.5 Durability Assessment

[0181] 2.2.11.5.1 Column robustness test

[0182] Based on the above-planned experimental conditions, the chromatographic columns were investigated using Shimadzu Shim-pack GIS C18 (2.1 mm × 150 mm, 2 μm), Shimadzu Shim-pack GIST C18 (2.1 mm × 150 mm, 2 μm), and Shimadzu Inertsil ODS-3 (2.1 mm × 150 mm, 2 μm). Figure 11 As shown in Table 5.

[0183] Table 5. Column robustness study - relative retention time ratio

[0184]

[0185] Figure 11 Different chromatographic columns were investigated; the results showed that this method is applicable to all three types of columns.

[0186] 2.2.11.5.2 Stability Assessment

[0187] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 6h, 8h, 12h, and 24h, as shown in Table 6.

[0188] Table 6 Stability Study - Retention Time

[0189]

[0190] The results showed that the RSD of the retention time of the characteristic peaks ranged from 0.63% to 1.10%, and the sample solution was stable within 24 hours. In summary, the RSD of the relative retention time of each characteristic peak met the requirements in the above investigations, indicating that the method is effective. The above six characteristic peaks will be included in subsequent investigations.

[0191] Example 1

[0192] Validation results of 3 batches of Jiao Liu Shen Qu formula granules

[0193] The proposed method was used to determine the characteristic spectra of three batches of this product, and the relative retention times were calculated. For example... Figure 12 As shown in Table 7. Figure 18 Comparative characteristic spectrum of Jiao Liu Shen Qu formula granules;

[0194] Table 7. Relative retention time of granules from three batches of Jiao Liu Shen Qu formula

[0195]

[0196] Figure 12 Formulation particle validation: Based on the principle that the relative retention time is stable and the peaks can be detected in all batches of samples and are relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks.

[0197] Example 2

[0198] Setting limits for relative retention time

[0199] Table 8 summarizes the methodological examination items and validation results:

[0200] Table 8 Summary of RSD% of Methodological Results—Relative Retention Time

[0201]

[0202] Therefore, the relative retention time of each peak is tentatively set at ±10%.

[0203] Example 3

[0204] Verification results of 23 batches of Liu Shen Qu medicinal materials

[0205] The characteristic spectra of 23 batches of medicinal materials were determined using the proposed method, and the relative retention times were calculated. See Table 9 for details.

[0206] The proposed method is to determine the substance by high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020).

[0207] Chromatographic conditions and system suitability tests are the same as in [Example 6 Content Determination].

[0208] Preparation of reference solution: Take appropriate amounts of uracil, adenine, and 5-hydroxymethylfurfural reference standards, accurately weigh them, and add 30% methanol to prepare a mixed solution containing 20µg of each per ml, which is used as the reference solution.

[0209] The preparation of the test solution is the same as in [Example 6 Content Determination].

[0210] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0211] The chromatogram of the test sample should show six characteristic peaks. Peaks 1, 4, and 5 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the adenine reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.64 (peak 2), 0.77 (peak 3), and 1.95 (peak 6).

[0212] See results Figure 19 , Figure 19 This is a comparative characteristic spectrum of charred Shenqu medicinal material.

[0213] Table 9. Relative retention times of 23 batches of medicinal materials

[0214]

[0215] Example 4

[0216] Verification results of 23 batches of Jiao Liu Shen Qu (a type of Chinese herbal medicine):

[0217] The proposed method is to determine the substance by high performance liquid chromatography (General Chapter 0512 of the Chinese Pharmacopoeia 2020).

[0218] Chromatographic conditions and system suitability tests are the same as in [Example 6 Content Determination].

[0219] Preparation of reference solution: Take appropriate amounts of uracil, adenine, and 5-hydroxymethylfurfural reference standards, accurately weigh them, and add 30% methanol to prepare a mixed solution containing 20µg of each per ml, which is used as the reference solution.

[0220] The preparation of the test solution is the same as in [Example 6 Content Determination].

[0221] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0222] The chromatogram of the test sample should show six characteristic peaks. Peaks 1, 4, and 5 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the adenine reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.64 (peak 2), 0.77 (peak 3), and 1.95 (peak 6).

[0223] The characteristic spectra of 23 batches of this product were determined using the proposed method, and the relative retention times were calculated. See Table 10 for details.

[0224] Table 10. Relative retention time of 23 batches of Jiao Liu Shen Qu medicinal slices

[0225]

[0226] Example 5

[0227] Verification results of 23 batches of Jiao Liu Shen Qu standard decoction

[0228] The characteristic spectra of 23 batches of this product were determined using the proposed method, and the relative retention times were calculated. The results are shown in Table 11.

[0229] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0230] Chromatographic conditions and system suitability tests are the same as in [Example 6 Content Determination].

[0231] Preparation of reference solution: Take appropriate amounts of uracil, adenine, and 5-hydroxymethylfurfural reference standards, accurately weigh them, and add 30% methanol to prepare a mixed solution containing 20µg of each per ml, which is used as the reference solution.

[0232] The preparation of the test solution is the same as in [Example 6 Content Determination].

[0233] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0234] The chromatogram of the test sample should show six characteristic peaks. Peaks 1, 4, and 5 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the adenine reference peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.64 (peak 2), 0.77 (peak 3), and 1.95 (peak 6).

[0235] The results are as follows Figure 20 , Figure 20 Comparative characteristic spectrum of standard decoction of Jiao Liu Shen Qu.

[0236] Table 11 Relative retention times of standard decoctions of Jiao Liu Shen Qu (a type of traditional Chinese medicine) from 23 batches

[0237]

[0238] Example 6 Content determination

[0239] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 2 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.20 mL / min; column temperature 25 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the adenine peak, should be no less than 3000.

[0240]

[0241] Preparation of the reference solution: Take an appropriate amount of adenine reference standard, accurately weigh it, and add 30% methanol to prepare a solution containing 20µg per ml.

[0242] Preparation of the test solution: Take an appropriate amount of Jiao Liu Shen Qu formula granules, grind them finely, take about 1.0 g, weigh accurately, place in a stoppered conical flask, accurately add 25 ml of 30% methanol, stopper tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 30% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0243] The assay involves precisely pipetting 1 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0244] 2.2.2.2 Selection of chromatographic conditions and system suitability assessment

[0245] 2.2.2.2.1 Determination of detection wavelength

[0246] Based on the above-planned experimental conditions, a diode array detector was used to perform a full-band scan of uridine and adenosine, as shown in the figure. Figure 13 . Figure 13 Ultraviolet absorption spectrum of adenine; as shown in the figure, the optimal detection wavelength for determining the adenine content in Jiao Liu Shen Qu granules was determined to be 260 nm through analysis of the spectrum.

[0247] 2.2.2.2.2 Column Temperature Investigation

[0248] Based on the above-planned experimental conditions, column temperatures of 25℃, 30℃, and 35℃ were investigated, with adenine separation rate and theoretical plate number used as evaluation indicators. See [link to relevant documentation]. Figure 14 Table 12. Figure 14 Column temperature test results.

[0249] Table 12 Analysis results at different column temperatures

[0250]

[0251] The results showed that the chromatographic peak resolution met the requirements at column temperatures of 20℃-30℃. Taking all factors into consideration, 25℃ was selected as the detection column temperature.

[0252] 2.2.2.2.3 Flow velocity investigation

[0253] Based on the above-established experimental conditions, flow rates of 0.2 ml / min, 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min were investigated, with adenine separation rate and theoretical plate number used as evaluation indicators. See [link to relevant documentation]. Figure 15 Table 13. Figure 15 Results of flow velocity study.

[0254] Table 13 Flow velocity investigation results

[0255]

[0256] The results showed that the target peaks could not be separated at 25℃ and 35℃, while the target peaks were well separated and symmetrical at 30℃. Therefore, the column temperature was finally determined to be 30℃.

[0257] 2.2.2.3 Investigation on the preparation of the test solution

[0258] 2.2.2.3.1 Examination of Extraction Methods

[0259] Take an appropriate amount of Jiao Liu Shen Qu formula granules, grind them into a fine powder, weigh about 1.0g accurately, place them in a stoppered conical flask, add 25ml of 30% methanol accurately, weigh again, sonicate (power 600W, frequency 40kHz), reflux for 30 minutes, cool, weigh again, replenish the lost weight with 30% methanol, shake well, filter, and collect the filtrate. Analyze and calculate the adenine content of different extraction methods. The results are shown in Table 14.

[0260] Table 14 Analysis results of different extraction methods

[0261]

[0262] The results showed that the results of ultrasonic extraction and reflux extraction were not significantly different, but ultrasonic extraction was simpler, more convenient and faster, so ultrasonic extraction was chosen.

[0263] 2.2.2.3.2 Investigation of Extraction Solvents

[0264] Take an appropriate amount of Jiao Liu Shen Qu (a traditional Chinese medicine formula) granules, grind them finely, and accurately weigh approximately 1.0 g. Place the weighing result in a stoppered conical flask. Accurately add 25 ml each of methanol, 30% methanol, 70% methanol, ethanol, dilute ethanol, and water, and weigh each flask. Sonicate the flask (600 W, 40 kHz) for 30 minutes, cool, and weigh again. Make up the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Calculate the adenine content extracted with different solvents. The results are shown in Table 15.

[0265] Table 15 Analysis results of different extraction solvents

[0266]

[0267] The results showed that when the extraction solvent was 30% methanol, the adenine content was relatively high. Therefore, 30% methanol was tentatively determined as the extraction solvent.

[0268] 2.2.2.3.3 Investigation of extraction time

[0269] Take an appropriate amount of the prepared Liuqu Shenqu formula granules, grind them finely, weigh accurately about 1.0 g, place them in a brown stoppered conical flask, accurately add 25 ml of 30% methanol, and ultrasonically treat them (power 600 W, frequency 40 kHz) for 15, 30, and 45 minutes respectively. Let it cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, and take the subsequent filtrate, thus obtaining the solution. Analyze and calculate the adenine content under different extraction times, as shown in Table 16.

[0270] Table 16 Analysis results of different extraction times

[0271]

[0272] The results showed that when the extraction time was 30 minutes, sufficient extraction could be achieved. Therefore, the extraction time of the test sample was determined to be 30 minutes.

[0273] 2.2.2.3.4 Investigation of solvent addition amount

[0274] Take an appropriate amount of the prepared Liuqu Shenqu formula granules, grind them finely, weigh accurately about 1.0 g, place them in a stoppered conical flask, accurately add 25, 50, and 100 ml of methanol respectively, ultrasonically treat them (power 600 W, frequency 40 kHz) for 30 minutes, let it cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, and take the subsequent filtrate, thus obtaining the solution. Investigate the extraction efficiency of the solvent addition amount. Calculate the adenine content, as shown in Table 17.

[0275] Table 17 Extraction efficiency of different solvent addition amounts

[0276]

[0277] The results showed that when the solvent addition amount was 25 ml, the extraction of the target components could be ensured to be complete. Therefore, the solvent addition amount was determined to be 25 ml.

[0278] 2.2.2.4 Methodology investigation

[0279] 2.2.2.4.1 Specificity experiment

[0280] Preparation of test sample solution: Prepare the test sample solution of Liuqu Shenqu granules according to the above-determined experimental conditions.

[0281] Preparation of reference substance solution: Weigh an appropriate amount of adenine reference substance accurately, and make a solution containing 20 μg per 1 ml with methanol, thus obtaining the solution.

[0282] Preparation of negative control solution: A negative control solution of pyrolysis-deficient Shenqu granules was prepared according to the experimental conditions outlined above. Results are shown below. Figure 16 . Figure 16 Specificity graph.

[0283] 2.2.2.4.2 Precision The reference solution was injected five times consecutively, and the peak area of ​​adenine was recorded. The RSD value was calculated, and the results are shown in Table 18.

[0284] Table 18 Precision Examination

[0285]

[0286] The results show that the precision is good.

[0287] 2.2.2.4.3 Linear Relationship

[0288] Take an appropriate amount of adenine and place it in a 50 ml volumetric flask. Dissolve it in methanol to prepare a series of linear solutions containing 37.45494 μg / ml adenine (purity 99.8%). Then dilute to concentrations of 1.4981976 μg / ml, 2.9963952 μg / ml, 7.490988 μg / ml, 14.981976 μg / ml, 22.472964 μg / ml, and 37.45494 μg / ml. Accurately pipette 2 μl of each solution into the liquid chromatograph and obtain the peak area. Plot the response curve with the injection concentration (X, mg / ml) on the x-axis and the peak area (Y) on the y-axis. The results are shown in Table 19 and Figure 17. Figure 17-1 Adenine standard curve. Figure 17-2 Uracil standard curve. Figure 17-3 This is a standard curve for 5-hydroxymethylfurfural.

[0289] Table 19-1 Results of Adenine Standard Curve Analysis

[0290]

[0291] The results showed that when the adenine concentration ranged from 1.4981976 to 37.45494 μg / ml, the linear relationship was y = 53.58x + 1.5138, with R² = 1. This indicates a good linear relationship within the concentration range of 1.4981976 to 37.45494 μg / ml.

[0292] Take appropriate amounts of uracil and 5-hydroxymethylfurfural, place them in a 50ml volumetric flask, and dissolve them in 30% methanol to prepare a solution containing 43.18565 μg / ml uracil (99.8% purity) and 89.70024 μg / ml 5-hydroxymethylfurfural (99.8% purity). Then dilute the solution. Accurately pipette 2μl of each solution into the liquid chromatograph and obtain the peak area. Plot a response curve with concentration (X, μg / ml) on the x-axis and peak area (Y) on the y-axis. The results are shown in Tables 19-1 and 19-2.

[0293] Table 19-2 Results of uracil standard curve analysis

[0294]

[0295] The results showed that the linear relationship of uracil was y = 20.483x + 9.2842 with R² = 0.9999 when the concentration range was 2.163664–43.27328 μg / ml. This indicates a good linear relationship within the concentration range of 2.163664–43.27328 μg / ml.

[0296] Table 19-3 Analytical Results of the Standard Curve for 5-Hydroxymethylfurfural

[0297]

[0298] The results showed that 5-hydroxymethylfurfural exhibited a linear relationship of y = 12.029x + 10.304 with R² = 0.9999 in the concentration range of 4.485012–89.70024 μg / ml. This indicates a good linear relationship within the concentration range of 4.485012–89.70024 μg / ml.

[0299] 2.2.2.4.4 Repeatability Experiment

[0300] Take approximately 1.0 g of the same test solution, accurately weigh 6 portions, and have the same operator prepare the test solution according to the established method. Calculate the adenine content of the 6 test solutions, and the results are shown in Table 20.

[0301] Table 20 Stability test results

[0302]

[0303] The results showed that the RSD value of adenine content obtained from the 6 repeatability tests was 1.0%, indicating that the method had good repeatability.

[0304] 2.2.2.4.5 Stability

[0305] Take about 1.0g of Jiao Liu Shen Qu formula granules, accurately weigh 6 portions, and have the same operator prepare the test solution according to the determined method. Calculate the content of the 6 test samples. The results are shown in Table 21.

[0306] Table 21 Results of Repeatability Experiments

[0307]

[0308] The results showed that the RSD values ​​of the adenine peak area were all 1.9%, and the test solution had good stability within 24 hours.

[0309] 2.2.2.4.6 Intermediate Precision

[0310] 2.2.2.4.6.1 Investigations by different personnel and at different times

[0311] The same sample was used, and different personnel (A and B) prepared the sample solution at different times (I and II) for analysis. The content of adenine in the sample was calculated, and the results are shown in Table 22.

[0312] Table 22 Results of intermediate precision experiments

[0313]

[0314] The results showed that the RSD value of adenine content was 1.1%, indicating good intermediate precision of this method.

[0315] 2.2.2.4.6.2 Investigation with different instruments

[0316] The test solution was prepared according to the determined method, and the results were investigated on an Agilent 1290 UHPLC, a Thermo Fisher HPLC, and a Thermo Fisher Vanquish Flex UHPLC (all columns were Shimadzu Shim-pack GIS C18, 2.1 mm × 150 mm, 2 μm). The adenine content was calculated, and the results are shown in Table 23.

[0317] Table 23 Experimental results with different instruments

[0318]

[0319] The results showed that the RSD value of the results measured by Shimadzu, Agilent 1290 and Thermo Fisher high performance liquid chromatographs was 3.3%, indicating that the instruments used in this method have good durability.

[0320] 2.2.2.4.7 Recovery rate

[0321] Take approximately 0.5 g of the test sample with a known content (adenine content 0.26 mg / g), make 6 portions, accurately weigh them, and accurately add 1 ml of 25 ml of adenine (purity 99.8%, concentration 81.69628 µg / ml) stock solution to each portion. Prepare and determine the test solution according to the prescribed method, calculate the recovery rate, and the results are shown in Table 24. The calculation formula is as follows:

[0322]

[0323] Table 24 Results of the sample recovery experiment

[0324]

[0325] The results showed that the average recovery rate of adenine was 2.1%, and the method had good accuracy.

[0326] 2.2.2.4.8 Durability Assessment

[0327] Based on the experimental conditions set above, the chromatographic columns of Shimadzu Shim-pack GIS C18 (2.1 mm × 150 mm, 2 μm), Shimadzu Shim-pack GIST C18 (2.1 mm × 150 mm, 2 μm), and Shimadzu Inertsil ODS-3 (2.1 mm × 150 mm, 2 μm) were investigated, and the results are shown in Table 25.

[0328] Table 25 Durability Test Results

[0329]

[0330] The results showed that the RSD% of adenine content was 3.3% among different chromatographic columns, indicating that this method is suitable for all three columns.

[0331] Example 6

[0332] Results of the content of 3 batches of tar-containing Shenqu:

[0333] Table 26 Test results of particle content in three batches of formulations

[0334]

[0335] Example 7

[0336] 23 batches of Liu Shen Qu (a traditional Chinese medicine) were tested, and the results are shown in Table 27:

[0337] Table 27 Results of the content of 21 batches of Shenqu medicinal materials

[0338]

[0339] Example 8

[0340] 23 batches of Jiao Liu Shen Qu (a type of medicinal herb) were tested, and the results are shown in Table 28:

[0341] Table 28 Results of the content of 23 batches of medicinal slices

[0342]

[0343] Example 9

[0344] 23 batches of Jiao Liu Shen Qu standard decoction were tested, and the results are shown in Table 29:

[0345] Table 29 Results of content determination in 23 batches of standard decoctions

[0346]

[0347] Comparative Example 1

[0348] Take 1g of Jiao Liu Shen Qu (a traditional Chinese medicine formula) granules, add 50mL of water, sonicate for 30min, filter, take 25mL of the filtrate, adjust the pH to 2-3 with dilute hydrochloric acid, extract twice with ethyl acetate, 25mL each time, combine the ethyl acetate extracts, evaporate to dryness in an 80℃ water bath, dissolve the residue in 5mL of 70% methanol, shake well, and take the filtrate. Inject 2μL of the test solution into the liquid chromatograph.

[0349] The method was as follows: High performance liquid chromatography (HPLC) was used for determination (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition). Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm); methanol was used as mobile phase A, and water as mobile phase B, with gradient elution as specified in the table below; the flow rate was 0.3 mL / min; the column temperature was 20 °C; the detection wavelength was 260 nm; and the theoretical plate number, calculated based on the guanosine peak, should not be less than 3000.

[0350]

[0351] The results are as follows Figure 21 The above, Figure 21 This is a diagram showing the results of the method in Comparative Example 1 of the present invention; by Figure 21 It can be seen that the method has poor separation. In addition, the method can only identify 5-hydroxymethylfurfural, but cannot identify uracil or adenine.

[0352] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic chromatograms of charred Shenqu (a type of medicinal herb), processed medicinal slices, formulated granules, and standard decoctions, comprising: The test sample was extracted with 30% methanol to obtain the test solution; The test solution was analyzed by HPLC to obtain the characteristic HPLC chromatograms of the charred Shenqu medicinal material, decoction pieces and standard decoction. Preparation of reference solutions: Uracil, adenine, and 5-hydroxymethylfurfural reference standards were dissolved in solvents to obtain reference solutions; The HPLC conditions were as follows: a C18 column; mobile phase A was acetonitrile solution, mobile phase B was 0.1% phosphoric acid aqueous solution, and gradient elution was used. The gradient elution specifically refers to: 0–10 min, Phase A: 1% → 5%, Phase B: 99% → 95%; 10–18 min, Phase A: 5% → 8%, Phase B: 95% → 92%; 18–25 min, Phase A: 8% → 14%, Phase B: 92% → 86%; 25–30 min, Phase A: 14%, Phase B: 86%; Detection wavelength: 260 nm; In the characteristic spectrum, peak 1 is uracil, peak 4 is adenine, and peak 5 is 5-hydroxymethylfurfural.

2. The method according to claim 1, characterized in that, The reference standard solution was injected into a high-performance liquid chromatograph for determination, and the chromatogram of the reference standard was obtained. Based on the characteristic parameters of the chromatogram of the reference standard, the corresponding chromatograms in the HPLC characteristic chromatograms of the scorched ginseng medicinal material, decoction pieces, standard decoction medicinal material test sample, standard decoction test sample, and formula granule test sample were compared and identified.

3. The method according to claim 2, characterized in that, The specific injection concentrations of the reference solutions were: uracil 20 μg / mL, adenine 20 μg / mL, and 5-hydroxymethylfurfural 20 μg / mL.

4. The method according to claim 1, characterized in that, The chromatographic column has the specifications of 150×2.1mm 2μm; the column temperature is 25℃; and the theoretical plate number, calculated based on adenine, should not be less than 3000.

5. The method according to claim 4, characterized in that, The flow rate of the mobile phase was 0.2 mL / min; the injection volume was 2 μL.

6. The method according to claim 4, characterized in that, The extraction method is ultrasonic extraction; The ultrasonic power was 600W, the frequency was 40kHz, and the extraction time was 30 minutes; the mass-to-volume ratio of the test sample raw material and 30% methanol was 1.0g:25mL.

7. The method according to claim 1, characterized in that, The similarity of HPLC characteristic chromatograms of charred Shenqu medicinal material, decoction pieces, and standard decoction was evaluated using a chromatographic fingerprint similarity evaluation system for traditional Chinese medicine. A standard HPLC characteristic chromatogram consisting of 12 characteristic peaks was constructed. The chemical composition information corresponding to each characteristic peak is as follows: peak 1 is uracil, peak 4 is adenine, and peak 5 is 5-hydroxymethylfurfural. The peak corresponding to the adenine reference standard peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values: the specified values ​​are: 0.64 for peak 2, 0.77 for peak 3, and 1.95 for peak 6.

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