Method for detecting quality of hedyotis diffusa formula granules for distinguishing counterfeit products from similar products

Abstract

The invention relates to a quality detection method for distinguishing counterfeit products from similar products of spreading hedyotis herb formula granules. The quality detection method comprises the following steps: carrying out thin-layer chromatography detection and high-performance liquid chromatography detection on the spreading hedyotis herb formula granules; the thin-layer chromatography detection comprises the following steps: sucking a test solution and a reference solution, respectively dripping the test solution and the reference solution on the same silica gel G thin-layer plate, developing by using a petroleum ether-toluene-acetic ether-glacial acetic acid solution as a developing solvent according to a volume ratio of petroleum ether to toluene to acetic ether to glacial acetic acid of 20: 40: 14: 1, taking out, airing, spraying a 10% ethanol sulfate solution, and detecting by using the thin-layer chromatography. Heating at 105 DEG C until the color of spots is clear, and viewing under an ultraviolet lamp; the high performance liquid chromatography detection method comprises the following steps: taking octadecylsilane chemically bonded silica as a filling agent; acetonitrile is used as a mobile phase A, a 0.1% phosphoric acid solution is used as a mobile phase B, and gradient elution is carried out; the flow rate is 0.8 ml per minute; the column temperature is 25 DEG C; according to the invention, the detection effect is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and in particular relates to a quality detection method for Hedyotis diffusa formula granules for distinguishing counterfeit products and similar products. Background Art

[0002] White flower snake tongue grass (Latin name: Scleromitrion diffusum), also known as: snake tongue grass, bamboo grass, fixed warp grass, dwarf white flower snake grass, etc. It is a plant of the genus Snake tongue grass in the Rubiaceae family and is a hairless herb.

[0003] Houttuynia cordata can be used as a medicinal material. According to the "Dictionary of Chinese Materia Medica", its functions and indications are: "clearing away heat and detoxifying, promoting blood circulation and reducing swelling, promoting dampness and reducing jaundice. It is mainly used to treat lung heat and cough, lung abscess, sore throat, intestinal abscess, boils, ulcers, snake bites, hot and painful stranguria, edema, dysentery, enteritis, damp-heat jaundice, and cancer"; Houttuynia cordata can also be made into cosmetic raw materials and is widely used in the cosmetics field; Houttuynia cordata is also often distilled and extracted to make Houttuynia cordata water carbonated beverages.

[0004] Waterline grass, also known as: corymb flower ear grass. Annual, loose, delicate herb, 15-50 cm high, bald or powdery hairs. Stems are branched, glabrous or sparsely covered with fine hairs. Leaves are opposite, sessile or with short petioles; leaf blades are linear-lanceolate, or linear, 1.5-3 cm long, 1.5-3.5 mm wide, with rough edges, often curled to the back; stipules are small, membranous, fused into sheaths, 1-1.5 mm long, truncated, with short prickles at the top. Inflorescences are axillary, mostly 2-5 flowers arranged in corymbs, rarely solitary; inflorescence stalks are linear, 0.6-2 cm long; calyx is broadly oval, about 2.5 mm long, with 4 teeth at the apex, and covered with fine hairs on the outside; corolla is funnel-shaped, white or light red, 2.5 mm long, with 4 lobes; 4 stamens; ovary is 2-chambered, style is linear, slightly extended, stigma is 2-lobed. The capsule is spherical, about 2.5 mm in diameter, with a flat top, splitting at the back of the chambers, and the calyx is persistent. The seeds are small and numerous. The flowering period is from July to September. The fruiting period is from September to October. This product is used as Hedyotis diffusa in Fujian, Guangdong, Guangxi and other places, and is a fake Hedyotis diffusa. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a quality detection method for Hedyotis diffusa formula granules for distinguishing counterfeit products and similar products, thereby improving the detection effect.

[0006] The embodiment of the present invention provides a quality detection method for Hedyotis diffusa formula granules for distinguishing counterfeit products and similar products, wherein the Hedyotis diffusa formula granules are subjected to thin layer chromatography detection and high performance liquid chromatography detection;

[0007] The steps of the thin layer chromatography detection are:

[0008] Take Hedyotis diffusa formula granules, grind them into powder, add ethanol, ultrasonicate them, cool them, filter them, evaporate the filtrate to dryness, add ethanol to dissolve the residue, and use it as the test solution;

[0009] Another reference medicinal material of Hedyotis diffusa was taken, water was added, decocted and kept slightly boiling, filtered, evaporated to dryness, and ethanol was added to the residue to obtain a reference medicinal material solution;

[0010] Take the above two solutions and spot them on the same silica gel G thin layer plate respectively, use petroleum ether-toluene-ethyl acetate-glacial acetic acid solution as the developing agent, the volume ratio of petroleum ether, toluene, ethyl acetate and glacial acetic acid is 20:40:14:1, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light;

[0011] The steps of the high performance liquid chromatography detection are:

[0012] Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the following table; the flow rate was 0.8 ml per minute; the column temperature was 25°C;

[0013]

[0014] In the high performance liquid chromatography detection, the preparation method of the reference medicinal material reference solution is to take the Hedyotis diffusa reference medicinal material, add methanol with a volume concentration of 80%, heat to reflux, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution;

[0015] Take an appropriate amount of cypermethrin reference substance, weigh it accurately, and add 30% methanol by volume to make a solution containing 100 μg per 1 ml.

[0016] The preparation method of the test solution is to take the Hedyotis diffusa formula granules, grind them into powder, add 30% methanol by volume, perform ultrasonic treatment, cool, shake well, filter, and obtain the filtrate.

[0017] Preferably, the preparation method of the Hedyotis diffusa formula granules is to take Hedyotis diffusa formula pieces, add water to decoct, filter, concentrate the filtrate into a clear paste, add auxiliary materials, dry, add auxiliary materials again, mix, granulate, and obtain Hedyotis diffusa formula granules.

[0018] Preferably, the high performance liquid chromatography method detects 10 characteristic peaks, of which three peaks correspond to compounds: deacetylcalciferol acid, deacetylcalciferol methyl ester and calciferol glycoside.

[0019] Preferably, when the thin layer chromatography method is used for detection, 0.5 g of Hedyotis diffusa formula granules are taken, ground into powder, and 20 mL of ethanol is added.

[0020] Preferably, during the thin layer chromatography detection, ethanol is added and then ultrasonic treatment is performed for 30 minutes.

[0021] Preferably, when the thin layer chromatography method is used for detection, 5 g of Hedyotis diffusa reference medicinal material is taken, 100 mL of water is added, the mixture is decocted and kept slightly boiling for 30 min, filtered, evaporated to dryness, and 10 mL of ethanol is added to the residue to obtain a reference medicinal material solution.

[0022] Preferably, in the HPLC detection, the filler size is: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0023] Preferably, in the HPLC detection, the preparation method of the control medicinal material reference solution is to take about 1.0 g of Hedyotis diffusa control medicinal material, place it in a stoppered conical flask, add 25 ml of 80% methanol by mass concentration, heat and reflux for 20 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

[0024] Preferably, in the HPLC detection, the preparation method of the test solution is to take 0.2 g of Hedyotis diffusa formula granules, grind them into powder, add 25 mL of 30% methanol by volume, ultrasonically treat for 20 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

[0025] Preferably, in the HPLC detection, 10 μl of the reference solution and the test solution are taken and injected into a liquid chromatograph for determination.

[0026] The beneficial effect of the present invention is that, through the method of the present invention, more characteristic peaks can be detected, and the separation between the peaks is good, which can well distinguish counterfeit products and similar products, and achieve high-quality detection of Hedyotis diffusa formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a reference characteristic spectrum of Example 1 of the present invention, wherein, Peak 1: deacetylcalciferol acid; Peak 3: deacetylcalciferol acid methyl ester; Peak 5 (S): calciferol acid; Chromatographic column: Shim-pack GIST C18-AQ, 4.6 mm×250 mm, 5 μm.

[0028] Figure 2 TLC spectrum for specificity investigation of Example 2: 1-negative control 2 μl; 2-control medicinal material 2 μl; 3-test sample 2 μl; 4-test sample 2 μl.

[0029] Figure 3 This is the TLC spectrum of Hedyotis diffusa formula granule samples at different temperatures. Figure 3 (A) is the TLC spectrum at 5°C. Figure 3(B) is the TLC spectrum under 35°C conditions, 1-negative control 2μl; 2-control drug 2μl; 3-test sample 2μl; 4-test sample 2μl.

[0030] Figure 4 This is the TLC spectrum of Hedyotis diffusa formula granule samples at different humidity levels. Figure 4 (A) is the TLC spectrum under 32% condition, Figure 4 (B) TLC spectrum under 58% condition, Figure 4 (C) is the TLC spectrum under 88% conditions. 1- Negative control 2 μl 2- Control drug 2 μl; 3- Test sample 2 μl; 4- Test sample 2 μl.

[0031] Figure 5 HPLC characteristic spectra of different extraction methods.

[0032] Figure 6 The HPLC characteristic spectra at different extraction times.

[0033] Figure 7 HPLC characteristic spectra of different extraction solvents.

[0034] Figure 8 The HPLC characteristic spectra of different sampling amounts.

[0035] Fig. 9 This is a comparison chart of specific properties.

[0036] Fig.10 Overlay characteristic spectra for repeatability experiments.

[0037] Fig.11 Overlay feature maps for precision testing.

[0038] Fig.12 This is a characteristic spectrum of common peak superposition for stability test.

[0039] Fig.13 Overlay characteristic spectra for different chromatographic columns (different manufacturers and different models).

[0040] Fig.14 This is an overlay of characteristic spectra of three batches of large-scale production samples.

[0041] Fig.15 It is an overlay of the characteristic spectra of the test sample, reference substance and reference medicinal material.

[0042] Fig.16 The common peak fitting diagram of three batches of large-scale production samples and control medicinal materials. Peak 1: deacetylcalendula acid; Peak 3: deacetylcalendula acid methyl ester; Peak 5 (S): calendula acid; Chromatographic column: Shim-pack GIST C18-AQ, 4.6mm×250mm, 5μm. DETAILED DESCRIPTION

[0043] Example 1

[0044] [Source] This product is a formula granule made from the dried whole herb of Hedyotisffusa Willd, a plant of the Rubiaceae family, which is processed according to the main quality indicators of standard decoctions.

[0045] [Preparation method] Take 5500g of Hedyotis diffusa slices, add water to boil, filter, concentrate the filtrate into a clear paste (the dry extract yield is 9.3% to 17.0%), add appropriate amount of auxiliary materials, dry (or dry and crush), add appropriate amount of auxiliary materials, mix well, granulate, and make 1000g.

[0046] [Properties] This product is brown to tan granules; slight odor, slightly bitter taste.

[0047] [Identification] Take 0.5g of this product, grind it, add 20ml of ethanol, ultrasonically treat it for 30 minutes, cool it, filter it, evaporate the filtrate, and add 1ml of ethanol to the residue to dissolve it as the test solution. Take another 5g of Hedyotis diffusa control medicinal material, add 100ml of water, decoct and keep it slightly boiling for 30 minutes, filter it, evaporate it, add 10ml of ethanol to the residue, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 2μl of each of the above two solutions and spot them on the same silica gel G thin layer plate, use petroleum ether (30-60℃)-toluene-ethyl acetate-glacial acetic acid (20:40:14:1) solution as the developing agent, develop it, take it out, dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and inspect it under ultraviolet light (365nm). In the test sample chromatogram, fluorescent spots of the same color appear at the corresponding positions in the control medicinal material chromatogram.

[0048] [Characteristic spectrum] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0049] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.8 ml per minute; the column temperature was 25°C; the detection wavelength was 254 nm. The theoretical plate number calculated based on the cypermethrin peak should be no less than 4000.

[0050]

[0051] Preparation of reference solution Take about 1.0g of Hedyotis diffusa reference medicinal material, place it in a stoppered conical flask, add 25ml of 80% methanol, heat and reflux for 20min, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of the reference substance of scutellaria baicalensis, accurately weigh it, and add 30% methanol to make a solution containing 100μg per 1ml.

[0052] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2 g, put it in a stoppered conical flask, add 25 ml of 30% methanol, ultrasonically treat it for 20 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the solution.

[0053] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0054] There should be 10 characteristic peaks in the chromatogram of the test sample, and the retention times should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, among which Peak 5 should correspond to the retention time of the reference material. The peak corresponding to the peak of the reference material is the S peak. The relative retention time of the remaining characteristic peaks and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.55 (peak 1), 0.73 (peak 2), 0.86 (peak 3), 0.92 (peak 4), 1.33 (peak 6), 1.60 (peak 7), 2.31 (peak 8), 2.41 (peak 9), 2.48 (peak 10).

[0055] [Inspection] It should comply with the relevant provisions under granules (General Rules 0104 of the 2020 Edition of the Chinese Pharmacopoeia).

[0056] [Extract] Determined by the hot infusion method under the alcohol-soluble extract determination method (General Rules 2201 of the 2020 edition of the Chinese Pharmacopoeia), using ethanol as the solvent, the content of which shall not be less than 14.0%.

[0057] [Content Determination] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0058] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 2 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.2 ml per minute; the column temperature was 30°C; the detection wavelength was 236 nm. The theoretical plate number calculated based on the cypermethrin peak should be no less than 6000.

[0059]

[0060] Preparation of reference solution: Take appropriate amount of deacetylcarbamic acid, carbamic acid glycoside, and deacetylcarbamic acid methyl ester reference substances, add 30% methanol to make a reference solution containing 50 μg of each per 1 ml, shake well, and obtain.

[0061] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 30% methanol, stopper it, weigh it, treat it with ultrasound for 20 minutes, cool it, weigh it again, make up the lost weight with 30% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0062] The determination method is to accurately pipette 1 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0063] This product contains 1g of scutellaria baicalenic acid (C 18 H 24 O 12 ), deacetyl oxalate methyl ester (C 17 H 24 O 11 ) and deacetylcarbamate (C 16 H 22 O 11 ) The total amount should be 4.0mg~18.0mg.

[0064] [Specifications] Each 1g of formula granules is equivalent to 5.5g of medicinal pieces.

[0065]

Storage

[0066] Example 2

[0067] Thin layer identification

[0068] Using Hedyotis diffusa as a control, petroleum ether (30-60°C)-toluene-ethyl acetate-glacial acetic acid (20:40:14:1) solution as a developing agent, and 10% sulfuric acid ethanol solution as a color developer, a thin layer identification method for this product was established. After methodological investigation and 3 batches of large-scale sample tests, the chromatographic spots of the test product were clear, and the negative control sample had no interference. The test process is as follows:

[0069] Test instruments, thin layer plates

[0070] Electronic balance (Mettler-Toledo Instrument Co., Ltd.); CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); silica gel G plate (Qingdao Ocean Silica Gel Desiccant Co., Ltd.); silica gel G plate (Yantai Huanghai Silica Gel Desiccant Co., Ltd.); silica gel G plate (Qingdao Hailang Silica Gel Desiccant Co., Ltd.).

[0071] Reference

[0072] Oldenlandia diffusa reference medicinal material (China Food and Drug Inspection Institute, batch number: 121117-201910, specification: 1g / bottle).

[0073] The method investigation is consistent with the method investigation of thin layer chromatography research data of Hedyotis diffusa formula granules and standard soup.

[0074] Exclusive inspection

[0075] According to the thin layer chromatography method, 2 μl of negative control solution, control medicinal material solution and test solution were respectively spotted on the same silica gel G thin layer plate, and petroleum ether (30-60°C)-toluene-ethyl acetate-glacial acetic acid (20:40:14:1) solution was used as the developing agent. The plate was developed, taken out, dried, sprayed with 10% ethanolic sulfuric acid, heated at 105°C until the spots were clear, and examined under ultraviolet light (365nm).

[0076] The results showed that the negative samples had no interference (see Figure 2 ).

[0077] Durability test

[0078] According to the proposed method, the influence of different temperature, different humidity and different thin layer chromatography plate conditions on the test results was investigated respectively.

[0079] Different temperature investigation

[0080] According to the thin layer chromatography test, 2 μl of the control medicinal material solution and the test sample solution were taken, and developed with petroleum ether (30-60°C)-toluene-ethyl acetate-glacial acetic acid (20:40:14:1) solution at 5°C and 35°C respectively. The samples were taken out, dried, sprayed with 10% ethanolic sulfuric acid, heated at 105°C until the spots were clear, and examined under ultraviolet light (365nm).

[0081] The results showed that when the temperature was 5℃ and 35℃, the chromatogram of the test sample showed fluorescent spots of the same color at the corresponding position of the chromatogram of the control medicinal material, and the spots were clear and the separation was good, indicating that the method is applicable in the relative temperature range of 5℃ to 35℃ and has good durability (see Figure 3 ).

[0082] Different humidity survey

[0083] According to the thin layer chromatography test, 2 μl of the control medicinal material solution and the test sample solution were taken and spotted on the same silica gel G thin layer plate, and petroleum ether (30-60°C)-toluene-ethyl acetate-glacial acetic acid (20:40:14:1) solution was used as the developing agent. The plate was developed at relative humidity of 32%, 58%, and 88%, respectively. The plate was taken out, dried, sprayed with 10% ethanolic sulfuric acid, heated at 105°C until the spots were clear, and observed under ultraviolet light (365nm) to observe the results.

[0084] The results showed that when the relative humidity was 32%, 58%, and 88%, the chromatogram of the test sample showed fluorescent spots of the same color at the corresponding positions of the chromatogram of the control medicinal material, and the spots were clear and the separation was good, indicating that the method was applicable in the relative humidity range of 32% to 88% and had good durability (see Figure 4 ).

[0085] summary

[0086] Based on the above considerations, the thin layer identification method of Hedyotis diffusa formula granules is determined as follows:

[0087] Take 0.5g of this product, grind it, add 20ml of ethanol, ultrasonically treat it for 30 minutes, cool it, filter it, evaporate the filtrate, and add 1ml of ethanol to the residue to dissolve it as the test solution. Take another 5g of Hedyotis diffusa control medicinal material, add 100ml of water, decoct and keep it slightly boiling for 30 minutes, filter it, evaporate it, add 10ml of ethanol to the residue, and prepare the control medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), take 2μl of the control medicinal material solution and the test solution, respectively, and spot them on the same silica gel G thin layer plate, use petroleum ether (30-60℃)-toluene-ethyl acetate-glacial acetic acid (20:40:14:1) solution as the developing agent, develop it, take it out, dry it, spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and inspect it under ultraviolet light (365nm). In the chromatogram of the test sample, a main fluorescent spot of the same color appears at the corresponding position in the chromatogram of the control medicinal material.

[0088] Example 3

[0089] Referring to the chromatographic conditions of the characteristic spectrum of the standard decoction, the reference medicinal material of Hedyotis diffusa and the reference substance of oxalis acid, the reference substance of deacetylated oxalis methyl ester and the reference substance of deacetylated oxalis acid were used as reference substances, and the characteristic spectrum of this product (presenting 10 characteristic peaks, of which the components of 3 peaks were identified) was established by high performance liquid chromatography [Shimadzu Shim-pack GIST C18-AQ (4.6mm×250mm, 5μm), acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution], and the results of the investigation of specificity, precision, repeatability, reproducibility, intermediate precision, stability and durability all met the requirements, so it was confirmed that this method was feasible. Its methodological investigation and verification are as follows:

[0090] Instruments, reagents and test drugs

[0091] (1) Instruments: Shimadzu HPLC (LC-2030 Plus, Shimadzu Co., Ltd., Japan); LabSolutions chromatography workstation, SPD-10A UV detector; Thermo Fisher (U3000, Thermo Fisher Scientific (China) Technology Co., Ltd.), Chromeleon 7.2 chromatography workstation, VWD-3x00 UV detector; Thermo Fisher (U3000RS, Thermo Fisher Scientific (China) Technology Co., Ltd.), Chromeleon 7.2 chromatography workstation, DAD-3x00 detector; Shimadzu Shim-pack GISTC18-AQ (4.6 mm × 250 mm, 5 μm); Mid-spectrum Blue XR-C18 (4.6 mm × 250 mm, 5 μm); Agilent 5TC C18

[0092] (2) (4.6 mm × 250 mm, 5 μm); Waters HSS T3 (4.6 mm × 250 mm, 5 μm); medium spectrum red AQ-C18 (4.6 mm × 250 mm, 5 μm); constant temperature water bath (HMTD-7000, Beijing Yongguangming Medical Instrument Co., Ltd.); ultrasonic cleaner (KQ-300DE, Kunshan Ultrasonic Instrument Co., Ltd.); one-tenth of a balance (PX224ZH, Ohaus Instrument Co., Ltd.); one-hundred-thousandth of a balance (AWU220D, Shimadzu Corporation of Japan).

[0093] (2) Reagents: ethanol (Tianjin Zhiyuan Chemical Reagent Co., Ltd.), methanol (Tianjin Komeo Chemical Reagent Co., Ltd.), acetonitrile (Tianjin Komeo Chemical Reagent Co., Ltd.) of chromatographic grade; water was ultrapure water (self-made in the laboratory); phosphoric acid (Tianjin Komeo Chemical Reagent Co., Ltd.); glacial acetic acid (Tianjin Zhiyuan Chemical Reagent Co., Ltd.); formic acid (Tianjin Komeo Chemical Reagent Co., Ltd.).

[0094] (3) Reference substances and reference medicinal materials: oxalicylic acid (batch number: DST200920-068, content: 98.54%, Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.), deacetyloxalicylic acid methyl ester (batch number: 111786-201602, content: 94.3%, China Food and Drug Inspection Institutes), deacetyloxalicylic acid (batch number: 3715, content: 95.7%, Shanghai Shidande Standard Technical Service Co., Ltd.), and Hedyotis diffusa reference medicinal material (batch number: 121183-201605, China Food and Drug Inspection Institutes).

[0095] Characteristic spectrum determination method

[0096] Chromatographic conditions and system suitability test. Octadecylsilane bonded silica gel was used as the filler (Shimadzu Shim-packGIST C18-AQ (4.6 mm × 250 mm, 5 μm)); acetonitrile was used as the mobile phase A, 0.1% phosphoric acid solution was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 0.8 ml per minute; the column temperature was 25°C; and the detection wavelength was 254 nm. The theoretical plate number calculated based on the cypermethrin peak should not be less than 4000.

[0097] Table 1 Gradient elution program

[0098]

[0099] Preparation of reference solution Take about 1.0g of Hedyotis diffusa reference medicinal material, place it in a stoppered conical flask, add 25ml of 80% methanol, heat and reflux for 20 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of scutellaria baicalensis reference substance, accurately weigh it, and add 30% methanol to make a solution containing 50μg per 1ml.

[0100] Preparation of test solution: Take an appropriate amount of this product (batch number: 220401), grind it into powder, take about 0.2 g, put it in a stoppered conical flask, add 25 ml of 30% methanol, ultrasonicate for 20 minutes, cool, shake well, filter, and obtain the filtrate.

[0101] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0102] Methodological investigation and verification of characteristic spectrum determination

[0103] The test solution was prepared by different extraction methods and tested according to the above test method. The results showed that the number of main peaks of the chromatogram of the test solution with different extraction methods was consistent, and the peak shape was not significantly different. However, the ultrasonic extraction effect was significantly better than the reflux method, so ultrasonic extraction was selected as the sample extraction method (see Figure 5 , Table 2).

[0104] Table 2 Comparison of extraction methods

[0105]

[0106] Extraction time investigation. The sample solutions were prepared by ultrasonic treatment at different times and tested according to the above test method. The results showed that the number of main peaks of the chromatograms of the sample solutions with different extraction times was consistent, and the peak shapes were not significantly different. However, the extraction effect of 20 minutes was better. In order to shorten the detection time, the extraction time of the sample was determined to be 20 minutes (see Table 3, Figure 6 ).

[0107] Table 3 Comparison of extraction time

[0108]

[0109] Investigation of extraction solvents. The test solutions were prepared with different extraction solvents and tested according to the above test method. The results showed that the extraction effect was best when the extraction solvent was 30% methanol, and the separation degree was high. Therefore, 30% methanol was determined as the extraction solvent (see Table 4, Figure 7 ).

[0110] Table 4 Comparison of extraction solvents

[0111]

[0112] The sample weight was investigated. The test solution was prepared with different sample weights and measured according to the above method. The results showed that the number of main peaks was consistent. When the sample weight was 0.2g, the extraction effect was the best. Therefore, the sample weight was determined to be 0.2g (see Table 5, Figure 8 ).

[0113] Table 5 Comparison of sampling volume

[0114]

[0115] In summary, the main parameters of the test solution preparation method are determined as follows: take an appropriate amount of the product, grind it into powder, take about 0.2 g, put it in a stoppered conical flask, add 25 ml of 30% methanol, ultrasonically treat it for 20 minutes, cool it, shake it well, filter it, and take the filtrate to obtain it.

[0116] Validation of the characteristic profile analysis method

[0117] Specificity investigation. Take the test sample and inject 10μl of maltodextrin according to the test sample treatment method, and measure according to the above chromatographic conditions. The test shows that the blank solvent has no interference (see Fig. 9 ).

[0118] 6.4.2 Repeatability test About 0.2g of Hedyotis diffusa formula granule sample (batch number: 220403) was taken, a total of 6 portions, and measured according to the method in item "6.2", with 6 injections for measurement. The measurement results showed that the peak shape and number of peaks in the characteristic spectrum of the test sample were basically consistent. There are 10 common peaks in the characteristic spectrum. Taking the herba sargentii peak (peak 5) as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10 and peak S were calculated, and the RSD value was calculated. The calculation results showed that the relative peak area RSD value and the relative retention time RSD value were both less than 3.0%, which were within the qualified range. The experiment showed that the method had good reproducibility (see Table 6, Table 7, Fig.10 )

[0119] Table 6 Relative retention time of characteristic spectra of repeatability test

[0120]

[0121] Table 7 Relative peak area of ​​repeatability test characteristic spectrum

[0122]

[0123] For the precision test, about 0.2 g of Hedyotis diffusa formula granules (batch number: 220403) were taken, and according to the test method, 6 injections were made continuously for determination. The test results showed that the peak shape and number of peaks in the characteristic spectrum of the test sample were basically consistent. There are 10 common peaks in the characteristic spectrum. The sarcosyl acid peak (peak 5) is used as the reference peak S. The relative retention time and relative peak area of ​​peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0%, which are within the qualified range. The test shows that the precision of the instrument is good (see Table 8, Table 9, Fig.11 )

[0124] Table 8 Precision test HPLC characteristic spectrum relative retention time

[0125]

[0126] Table 9 Precision test HPLC characteristic spectrum relative peak area

[0127]

[0128] Stability test: Take a batch of samples of about 0.2g, and according to the test method, inject and measure at 0h, 2h, 4h, 8h, 12h, and 24h respectively. The peak shape and peak number are basically stable (see Fig.12 ), there are 10 common peaks in the characteristic spectrum, with the cyperus erythritol peak (peak 5) as the reference peak S, and the relative retention time and relative peak area of ​​peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0%, which are within the qualified range (see Table 10 and Table 11), indicating that the test solution is relatively stable within 24 hours.

[0129] Table 10 Relative retention time of characteristic spectrum of stability test

[0130]

[0131]

[0132] Table 11 Relative peak area of ​​characteristic spectrum of stability test

[0133]

[0134] Intermediate precision inspection

[0135] Other analysts of this project operated on different dates and different equipment, took about 0.2g of the same batch of Hedyotis diffusa formula granules (batch number: 220403), accurately weighed, and 6 parallel portions were used to prepare the test solution according to the method and analyzed. There are 10 common peaks in the characteristic spectrum. The peak of scutellaria baicalensis (peak 5) is used as the reference peak S. The relative retention time and relative peak area of ​​peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative peak areas are all less than 3% and the relative retention times are all within ±10% (see Table 12-Table 15). The experiment shows that the intermediate precision of this method is good.

[0136] Table 12 Precision relative retention time of characteristic spectrum of Hedyotis diffusa formula granules

[0137]

[0138]

[0139] Table 13 Precision relative peak area of ​​characteristic spectrum of Hedyotis diffusa formula granules

[0140]

[0141]

[0142] Note: Operation time: July 28, 2022; Chromatograph: Shimadzu LC-2030Plus; Equipment number: E01-630; Operation time: August 17, 2022; Chromatograph: Thermo Fisher U3000RS; Equipment number: E01-642.

[0143] Table 14 Relative retention time of characteristic spectrum repeatability of Hedyotis diffusa formula granules

[0144]

[0145] Table 15 Relative peak area of ​​characteristic spectrum repeatability of Hedyotis diffusa formula granules

[0146]

[0147]

[0148] Note: Operation time: July 27, 2022; Chromatograph: Shimadzu LC-2030Plus; Equipment number: E01-630;

[0149] Operation time: August 17, 2022; Chromatograph: Thermo Fisher U3000RS; Equipment number: E01-642.

[0150] Reproducibility test. Take about 0.2g of Hedyotis diffusa formula granules (batch number: 220403), a total of 6 samples, and measure according to the method, and inject 6 needles for measurement. The measurement results show that the peak shape and number of peaks of the characteristic spectrum of the test sample are basically the same. There are 10 common peaks in the characteristic spectrum. The scutellaria baicalensis glycoside peak (peak 5) is used as the reference peak S. The relative retention time and relative peak area of ​​peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10 and peak S are calculated, and the RSD value is calculated. The calculation results show that the relative peak area RSD value and the relative retention time RSD value are both less than 3.0%, which are within the qualified range. The experiment shows that the method has good reproducibility (see Table 16 and Table 17).

[0151] Table 16 Relative retention time of characteristic spectrum of reproducibility test

[0152]

[0153]

[0154] Table 17 Relative peak area of ​​characteristic spectrum of reproducibility test

[0155]

[0156]

[0157] Operation time: August 17, 2022; Chromatograph: Shimadzu LC-2030Plus; Equipment number: E01-642;

[0158] Operation time: July 28, 2022; Chromatograph: Thermo Fisher U3000RS; Equipment number: E01-630;

[0159] Durability test

[0160] Investigation of different chromatographic columns (different manufacturers, different models) About 0.2 g of Hedyotis diffusa formula granules (batch number: 220403) were taken and chromatographically analyzed using three chromatographic columns (4.6 mm × 250 mm, 5 μm) of different models and manufacturers according to the test method. C18 (PF-95), Shimadzu GIST AQ-C18 (CX-135), and Medium Spectrum Blue XR-C18) were injected and tested. The test results showed that the peak shape and peak number of the characteristic spectrum of the test samples were inconsistent (see Fig.13). There are 10 common peaks in the characteristic spectrum. Taking the cypermethrin peak (peak 5) as the reference peak S, the relative retention time and relative peak area of ​​peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, peak 10 and peak S are calculated, and the RSD% value is calculated. The calculation results show that the relative retention time RSD% value and the relative peak area RSD value are greater than 2.0% and greater than 3.0%, respectively, both within the unqualified range (see Table 18 and Table 19). The experiment shows that different models of chromatographic columns from different manufacturers have a great influence on the determination of characteristic spectra.

[0161] Table 18 Relative retention time of characteristic spectra of different chromatographic columns (different manufacturers and models)

[0162]

[0163] Table 19 Relative peak areas of characteristic spectra of different chromatographic columns (different manufacturers, different models)

[0164]

[0165]

[0166] summary

[0167] The characteristic spectrum method was tested for specificity, precision, repeatability, reproducibility, intermediate precision, stability and durability, and the results all met the requirements, indicating that the established method can be well used for the characteristic spectrum determination of Hedyotis diffusa formula granules.

[0168] Characterization and analysis of characteristic spectra of three batches of finished products

[0169] Determination of characteristic spectra of three batches of finished products. According to the characteristic spectrum analysis method proposed above, the characteristic chromatograms of three batches of large-scale production samples were determined. The results showed that there were 10 common peaks in the characteristic chromatograms of the three batches of large-scale production samples, and the retention times of the 10 characteristic peaks in the chromatograms of the reference medicinal material reference substances corresponded to each other. Among them, peak 5 corresponded to the retention time of the peak of the reference substance of the herbal glycoside reference substance. The common peak characteristic spectrum (see Fig.14 — Fig.16 ).

[0170] Evaluation of characteristic chromatograms of three batches of large-scale production The characteristic spectra of three batches of large-scale production samples were evaluated for similarity, and 10 common characteristic peaks were calibrated, among which peak 5 was the characteristic peak of the ceramide reference substance. The peak corresponding to the ceramide reference substance was taken as the S peak, and the relative retention times of characteristic peaks peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, peak 9, and peak 10 were calculated. The relative retention times and ranges are shown in (Table 24).

[0171] Table 24 Relative retention time of characteristic spectra of three batches of production samples

[0172]

[0173] In summary, the characteristic spectrum detection method of the formula granules of this product was established by high performance liquid chromatography. The characteristic spectrum of three batches of scale production samples was evaluated, and 10 common characteristic peaks were calibrated, among which peak 5 corresponded to the retention time of the reference peak of the cypermethrin acid reference substance, and the peak corresponding to the cypermethrin acid reference substance was taken as the S peak. The relative retention time of peaks 1, 2, 3, 4, 6, 7, 8, 9, 10 and S peak was calculated, and the average relative retention time of the characteristic peaks of three batches of scale production samples was proposed as the specified value, which were: 0.55 (peak 1), 0.73 (peak 2), 0.86 (peak 3), 0.92 (peak 4), 1.33 (peak 6), 1.60 (peak 7), 2.31 (peak 8), 2.41 (peak 9), 2.48 (peak 10). Combined with the results of standard decoction, small-scale test and scale production test and experimental error, the relative retention time allowable range of each peak was proposed to be ±10%.

[0174] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0175] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. A quality detection method for distinguishing fake and similar products of Hedyotis diffusa formula granules, characterized in that: The Hedyotis diffusa formula granules were tested by thin layer chromatography and high performance liquid chromatography; The steps of the thin layer chromatography detection are: Take the Hedyotis diffusa formula granules, grind them into powder, add ethanol, ultrasonicate them, cool them, filter them, evaporate the filtrate to dryness, add ethanol to dissolve the residue, and use it as the test solution; Another reference medicinal material of Hedyotis diffusa was taken, water was added, decocted and kept slightly boiling, filtered, evaporated to dryness, and ethanol was added to the residue to obtain a reference medicinal material solution; Take the above two solutions and spot them on the same silica gel G thin layer plate respectively, use petroleum ether-toluene-ethyl acetate-glacial acetic acid solution as the developing agent, the volume ratio of petroleum ether, toluene, ethyl acetate and glacial acetic acid is 20:40:14:1, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and examine under ultraviolet light; The steps of the high performance liquid chromatography detection are: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in the following table; the flow rate was 0.8 ml per minute; the column temperature was 25°C; In the high performance liquid chromatography detection, the preparation method of the reference medicinal material reference solution is to take the Hedyotis diffusa reference medicinal material, add methanol with a volume concentration of 80%, heat to reflux, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution; Take an appropriate amount of cypermethrin reference substance, weigh it accurately, and add 30% methanol by volume to make a solution containing 100 μg per 1 ml. The preparation method of the test solution is to take the Hedyotis diffusa formula granules, grind them into powder, add 30% methanol by volume, perform ultrasonic treatment, cool, shake well, filter, and obtain the filtrate.

2. The detection method according to claim 1, characterized in that: The preparation method of the Hedyotis diffusa formula granules comprises the following steps: taking Hedyotis diffusa formula pieces, adding water to boil, filtering, concentrating the filtrate into a clear paste, adding auxiliary materials, drying, adding auxiliary materials again, mixing, granulating, and obtaining the Hedyotis diffusa formula granules.

3. The detection method according to claim 1, characterized in that: The high performance liquid chromatography method detects 10 characteristic peaks, of which three peaks correspond to compounds: deacetylcalciferol acid, deacetylcalciferol methyl ester and calciferol glycoside.

4. The detection method according to claim 1, characterized in that: When the thin layer chromatography method is used for detection, 0.5 g of Hedyotis diffusa granules are taken, ground into powder, and 20 mL of ethanol is added.

5. The detection method according to claim 1, characterized in that: When the thin layer chromatography method is used for detection, ethanol is added and then ultrasonic treatment is performed for 30 minutes.

6. The detection method according to claim 1, characterized in that: When the thin layer chromatography method is used for detection, 5 g of Hedyotis diffusa reference medicinal material is taken, 100 mL of water is added, the mixture is decocted and kept slightly boiling for 30 min, filtered, evaporated to dryness, and 10 mL of ethanol is added to the residue to obtain a reference medicinal material solution.

7. The detection method according to claim 1, characterized in that: In the HPLC detection, the filler size is: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

8. The detection method according to claim 1, characterized in that: In the high performance liquid chromatography detection, the preparation method of the control medicinal material reference solution is to take about 1.0 g of Hedyotis diffusa control medicinal material, place it in a stoppered conical flask, add 25 ml of 80% methanol by mass concentration, heat and reflux for 20 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution.

9. The detection method according to claim 1, characterized in that: In the high performance liquid chromatography detection, the preparation method of the test solution is to take 0.2 g of Hedyotis diffusa formula granules, grind them into powder, add 25 mL of 30% methanol by volume, ultrasonically treat for 20 minutes, cool, shake well, filter, and take the filtrate to obtain the solution.

10. The detection method according to claim 1, characterized in that: In the HPLC detection, 10 μl of the reference solution and the test solution were taken and injected into the liquid chromatograph for determination.

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