Determination method of component content of Dermatosis Xuedu Pills, construction method of fingerprint spectrum and its application

Abstract

The present invention provides a method for determining the content of components of a skin disease blood poison pill, a method for constructing a fingerprint spectrum and its application, and relates to the technical field of quality detection methods for traditional Chinese medicine preparations. The determination method comprises the following steps: weighing a reference substance, mixing it with a solvent, and preparing a mixed reference substance solution; preparing a test solution; drawing the reference substance solution and the test solution, respectively injecting them into a liquid chromatograph, determining, and obtaining a chromatogram; the two methods use the same high-performance liquid chromatography conditions and are applied to the quality control of skin disease blood poison pill preparations, which is faster and more efficient than using two methods to detect fingerprints and contents respectively. Not only the contents of 7 components of gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol and rubiacein in the skin disease blood poison pill were determined, but also the constructed fingerprint spectrum calibrated 54 peaks. This method can more comprehensively and systematically reflect the quality of the skin disease blood poison pill than the determination of the content of a single component.

Description

Technical Field

[0001] The invention relates to the technical field of quality detection methods for traditional Chinese medicine preparations, and in particular to a method for determining the content of components of a dermatopathy blood poison pill, a method for constructing a fingerprint spectrum and applications thereof. Background Art

[0002] The Skin Disease Blood Poison Pill is a prescription made by the famous modern doctor Shi Jinmo. It is made of madder, peach kernel, schizonepeta spicate (charcoal), snake skin (fried with wine), red peony root, angelica, Imperata root, Kochia fruit, Xanthium sibiricum fruit (fried), Rehmannia root, Forsythia suspensa, Honeysuckle, Kuyeding, Smilax glabra, Phellodendron amurense, Gleditsia sinensis thorn, Platycodon grandiflorum, Leonurus japonicus, bitter almond (peeled and fried), Saposhnikovia divaricata, red Poria, white peony root, cicada skin, burdock fruit (fried), peony bark, Dictamni It is composed of 39 ingredients, including angelica root, rehmannia root, rhubarb (fried with wine), honeysuckle vine, lithospermum officinale, fritillaria thunbergii, ligusticum chuanxiong (roasted with wine), licorice, angelica dahurica, amaranth seed, bauhinia bark, millettia reticulata, duckweed, and safflower. It has the effects of clearing blood, detoxifying, reducing swelling and relieving itching. It is used for rashes, eczema, itchy skin, freckles and acne, red face and nose, sores, scabies, dizziness, and dry stool caused by disharmony of meridians, dampness, heat and dryness of blood.

[0003] Due to the complexity of Chinese medicine ingredients and the integrity of Chinese medicine effects, it is often not a single ingredient that plays a pharmacological role. Using a single ingredient or several ingredients as quality control indicators is increasingly incompatible with the requirements of Chinese medicine quality control. Therefore, Chinese medicine fingerprint technology came into being. Chinese medicine fingerprint technology originated from fingerprint identification, and uses modern information technology and quality analysis methods to more comprehensively reflect the types and quantities of chemical components contained in Chinese medicine. For Chinese medicinal materials, fingerprints can be used to identify authenticity and judge the quality; for Chinese patent medicines, fingerprints can identify product authenticity, judge the rationality of the preparation process, and effectively control product quality. At this stage, most of the effective ingredients of Chinese medicine are still unclear. The integrity and ambiguity of Chinese medicine fingerprints are just adapted to this feature, and are more scientific and comprehensive than the quality control method of a single ingredient. At present, Chinese medicine fingerprints have been recognized internationally as a control mode for Chinese medicine quality. High performance liquid chromatography has the advantages of high separation efficiency and fast analysis speed, and has become the main analytical method for fingerprints today.

[0004] The quality standard for Dermatology Xuedu Pills (standard number WS3-B-1320-93-2006, WS3-B1320-2002) has only thin layer identification items for Phellodendron chinense and Rhubarb under the identification items, but no content determination items, which cannot meet the requirements of comprehensive quality control. Literature search found that the article "HPLC determination of the content of 7 ingredients in Dermatosis Blood Toxin Tablets" determined the content of chlorogenic acid, hydroxysafflorchrome A, paeoniflorin, forsythiaside A, ferulic acid, berberine and glycyrrhizic acid in Dermatosis Blood Toxin Tablets; the article "HPLC fingerprint study of Dermatosis Blood Toxin Tablets and content determination" established a fingerprint that calibrated 42 common peaks, the separation of the 42 common peaks was poor, and the common peaks were not identified. At the same time, the content of chlorogenic acid, berberine and glycyrrhizic acid was determined; the article "HPLC fingerprint study of Dermatosis Blood Toxin Pills" established a fingerprint that calibrated 20 common peaks and identified the corresponding components of 8 of the common peaks. The prescription of Dermatosis Blood Toxin Pills is composed of 39 herbs with complex ingredients. The existing literature cannot fully reflect the product quality of Dermatosis Blood Toxin Pills. Therefore, it is very necessary to establish a detection method that can fully reflect the product quality of Dermatosis Blood Toxin Pills.

[0005] Based on this, the present invention provides a detection method that more comprehensively reflects the product quality of the Dermatological Blood Toxin Pills. A comprehensive fingerprint detection method is established, and the constructed fingerprint is used to calibrate 54 common peaks, and the attribution of the common peaks is analyzed to determine the source of each common peak; 14 common peaks are identified by reference substances to determine the names of their corresponding chemical components; the present invention simultaneously determines the contents of 7 components, namely, gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol and rubiacein, and provides quantitative indicators of the 7 components; the fingerprint detection method provided by the present invention and the quality control of the content determination of the 7 components are integrated into one detection method, which is fast and efficient, and has important significance for the comprehensive quality detection and overall quality control of the Dermatological Blood Toxin Pills. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for determining the component content of skin disease blood poison pills and a method for constructing a fingerprint spectrum. By optimizing the extraction solvent, extraction time, selecting the mobile phase, mobile phase elution program, chromatographic column and other conditions, it is ensured that the effective ingredients in the skin disease blood poison pills are extracted to the greatest extent, and more comprehensive effective ingredient information is obtained.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] On the one hand, the present invention provides a method for determining the content of components of Dermatology Xuedu Pills, wherein the components include gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol and rubiacein, comprising the following steps:

[0009] S1: Preparation of reference solution: weigh reference substances gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol, and rubiacein, and mix them with a solvent to prepare a mixed reference solution;

[0010] S2: Prepare the test solution: weigh the skin disease blood poison pills, mix with the solvent, weigh to determine the weight, extract, cool, weigh again, make up the lost weight with the solvent, shake well, filter, and take the filtrate;

[0011] S3: Pipette the reference solution and the test solution, inject them into the liquid chromatograph respectively, measure and obtain the chromatogram;

[0012] The chromatographic conditions are:

[0013] Chromatographic column: non-polar reverse phase chromatography column;

[0014] Mobile phase: acetonitrile solution is mobile phase A, and 0.05%-0.15% phosphoric acid aqueous solution is mobile phase B;

[0015] Detection wavelength: 225-235nm;

[0016] Flow rate: 0.9-1.1 mL min -1 ;

[0017] Column temperature: 25℃-35℃;

[0018] The elution method is gradient elution, and the elution gradient is:

[0019] 0-35min, 2%-23% mobile phase A, 98%-77% mobile phase B;

[0020] 35-70 min, 23%-80% mobile phase A, 77%-20% mobile phase B;

[0021] 70-80min, 80%-90% mobile phase A, 20%-10% mobile phase B.

[0022] Preferably, the solvent described in S1 is methanol;

[0023] Preferably, the concentration of each reference substance in the mixed reference solution described in S1 is: gallic acid 7-9 μg / mL, chlorogenic acid 18-22 μg / mL, paeoniflorin 28-32 μg / mL, berberine hydrochloride 5-7 μg / mL, arctiin 8-10 μg / mL, paeonol 4-6 μg / mL, and madderin 3-5 μg / mL. Further preferably, the concentration of each reference substance in the mixed reference solution described in S1 is: gallic acid 7-8 μg / mL, chlorogenic acid 20-21 μg / mL, paeoniflorin 30-31 μg / mL, berberine hydrochloride 6-7 μg / mL, arctiin 8-9 μg / mL, paeonol 4-5 μg / mL, and madderin 3-4 μg / mL.

[0024] Preferably, the solvent described in S2 is an aqueous solution of methanol; further preferably, the volume fraction of methanol in the aqueous solution of methanol is 60%-80%, and most preferably, the volume fraction of methanol in the aqueous solution of methanol is 80%.

[0025] Preferably, the ultrasonic extraction time in S2 is 30-60 min, the ultrasonic power is 450-600 W, and the frequency is 35-45 kHz; further preferably, the ultrasonic extraction time in S2 is 30 min, the ultrasonic power is 500 W, and the frequency is 40 kHz.

[0026] Preferably, the concentration of the test solution described in S2 is 18-40 g / L; further preferably, the concentration of the test solution described in S2 is 20 g / L.

[0027] Preferably, the mass of the sample weighed in S2 is 1 g.

[0028] Preferably, the volumes of the reference solution and the test solution absorbed in S3 are 10 μL each.

[0029] Preferably,

[0030] The chromatographic conditions are:

[0031] Column: Platisil ODS, 5um, 4.6×250mm;

[0032] Mobile phase: acetonitrile solution is mobile phase A, and 0.05-0.1% phosphoric acid aqueous solution is mobile phase B;

[0033] Detection wavelength: 225-230nm;

[0034] Flow rate: 0.9-1 mL min -1 ;

[0035] Column temperature: 25-30℃;

[0036] The elution method is gradient elution, and the elution gradient is:

[0037] 0-15 min, 2%-14% mobile phase A, 98%-86% mobile phase B;

[0038] 15-35 min, 14%-23% mobile phase A, 86%-77% mobile phase B;

[0039] 35-70 min, 23%-80% mobile phase A, 77%-20% mobile phase B;

[0040] 70-80min, 80%-90% mobile phase A, 20%-10% mobile phase B.

[0041] Further preferably, the chromatographic conditions are:

[0042] Column: Platisil ODS, 5um, 4.6×250mm;

[0043] Mobile phase: acetonitrile solution is mobile phase A, and 0.1% phosphoric acid aqueous solution is mobile phase B;

[0044] Detection wavelength: 230nm;

[0045] Flow rate: 1 mL min -1 ;

[0046] Column temperature: 30°C;

[0047] The elution method is gradient elution, and the elution gradient is:

[0048] Time / min Mobile phase A / % Mobile phase B / % 0 2 98 8 10 90 15 14 86 20 16 84 35 23 77 50 40 60 60 70 30 70 80 20 80 90 10

[0049] In another aspect, the present invention provides a method for constructing a fingerprint, comprising the following steps:

[0050] S1: Prepare reference substance solution: weigh reference substances such as gallic acid, chlorogenic acid, hydroxysafflor yellow A, paeoniflorin, forsythiaside A, ferulic acid, berberine hydrochloride, arctiin, glycyrrhizic acid, paeonol, emodin, chrysophanol, physophanol methyl ether, and rubiacin, respectively, and mix them with solvent to obtain reference substance solution;

[0051] S2: Prepare the test solution: weigh the sample, mix it with the solvent, extract it, weigh it, cool it, weigh it again, make up the lost weight with the solvent, shake it well, filter it, and take the filtrate to obtain the solution;

[0052] S3: Prepare negative test solution: Weigh madder, red peony root and white peony root, Chinese angelica and Chuanxiong, Xanthium sibiricum, honeysuckle and honeysuckle, forsythia, phellodendron, bitter almond and peach kernel, arctium, peony bark, rhubarb, liquorice, safflower, and prepare negative test solution according to the steps of S2;

[0053] S4: respectively aspirate the reference solution, the test solution, and the negative test solution, and inject them into the liquid chromatograph, respectively, and measure to obtain a chromatogram;

[0054] The chromatographic conditions are:

[0055] Chromatographic column: non-polar reverse phase chromatography column;

[0056] Mobile phase: acetonitrile solution is mobile phase A, and 0.05%-0.15% phosphoric acid aqueous solution is mobile phase B;

[0057] Detection wavelength: 225-235nm;

[0058] Flow rate: 0.9-1.1 mL min -1 ;

[0059] Column temperature: 25℃-35℃;

[0060] The elution method is gradient elution, and the elution gradient is:

[0061] 0-35min, 2%-23% mobile phase A, 98%-77% mobile phase B;

[0062] 35-70 min, 23%-80% mobile phase A, 77%-20% mobile phase B;

[0063] 70-80min, 80%-90% mobile phase A, 20%-10% mobile phase B.

[0064] Preferably, the solvent described in S1 is methanol;

[0065] Preferably, the concentration of the reference solution described in S1 is 0.08-0.12 mg / mL.

[0066] Preferably, the solvent described in S2 is methanol; further preferably, the volume fraction of the methanol is 60%-80%, and most preferably, the volume fraction of the methanol is 80%.

[0067] Preferably, the ultrasonic extraction time in S2 is 30-60 min, the ultrasonic power is 450-600 W, and the frequency is 35-45 kHz; further preferably, the ultrasonic extraction time in S2 is 30 min, the ultrasonic power is 500 W, and the frequency is 40 kHz.

[0068] Preferably, the concentration of the test solution described in S2 is 18-40 g / L; further preferably, the concentration of the test solution described in S2 is 20 g / L.

[0069] Preferably, the mass of the sample weighed in S2 is 1 g.

[0070] Preferably, the volumes of the reference solution and the test solution absorbed in S3 are 10 μL each.

[0071] Preferably,

[0072] The chromatographic conditions are:

[0073] Column: Platisil ODS, 5um, 4.6×250mm;

[0074] Mobile phase: acetonitrile solution is mobile phase A, and 0.05-0.1% phosphoric acid aqueous solution is mobile phase B;

[0075] Detection wavelength: 225-230nm;

[0076] Flow rate: 0.9-1 mL min -1 ;

[0077] Column temperature: 25-30℃;

[0078] The elution method is gradient elution, and the elution gradient is:

[0079] 0-15 min, 2%-14% mobile phase A, 98%-86% mobile phase B;

[0080] 15-35 min, 14%-23% mobile phase A, 86%-77% mobile phase B;

[0081] 35-70 min, 23%-80% mobile phase A, 77%-20% mobile phase B;

[0082] 70-80min, 80%-90% mobile phase A, 20%-10% mobile phase B.

[0083] Further preferably, the chromatographic conditions are:

[0084] Column: Platisil ODS, 5um, 4.6×250mm;

[0085] Mobile phase: acetonitrile solution is mobile phase A, and 0.1% phosphoric acid aqueous solution is mobile phase B;

[0086] Detection wavelength: 230nm;

[0087] Flow rate: 1 mL min -1 ;

[0088] Column temperature: 30°C;

[0089] The elution method is gradient elution, and the elution gradient is:

[0090]

[0091]

[0092] On the other hand, the present invention provides the application of the method for determining the content of ingredients in the Skin Disease Blood Toxin Pills or the method for constructing the fingerprint spectrum in the quality control of the Skin Disease Blood Toxin Pills preparations.

[0093] Compared with the prior art, the present invention has the following beneficial effects:

[0094] 1. The fingerprint spectrum and multi-component content determination method of the skin disease blood poison pill provided by the present invention include the construction of the fingerprint spectrum method and the multi-component content determination method, using the same high performance liquid chromatography conditions and the same test sample solution preparation method, and the collected test sample chromatograms are used for fingerprint spectrum evaluation and content calculation, respectively, which is faster and more efficient than using two methods to detect fingerprint spectrum and content separately.

[0095] 2. The fingerprint spectrum and multi-component content determination method of the Skin Disease Blood Toxin Pill provided by the present invention take the Skin Disease Blood Toxin Pill as the detection object, optimize the extraction solvent and extraction time through multiple experiments, ensure that the effective ingredients in the Skin Disease Blood Toxin Pill are extracted to the maximum extent, and obtain more comprehensive effective ingredient information; optimize the selection of mobile phase, mobile phase elution program, chromatographic column and other conditions through multiple experiments to ensure that the effective ingredients in the Skin Disease Blood Toxin Pill are well separated, and the peak shape is good and there is no interference.

[0096] 3. The fingerprint spectrum and multi-component content determination method of the Dermatosis Blood Poison Pill provided by the present invention overcome the defect that the quality of the Dermatosis Blood Poison Pill cannot be comprehensively and clearly detected due to the interference caused by the complex chemical components of traditional Chinese medicine under the preferred chromatographic conditions. The Dermatosis Blood Poison Pill fingerprint spectrum calibrates 54 common peaks, and the chemical names of 14 of them are identified with reference to the reference substance, namely, peak 5 gallic acid, peak 11 chlorogenic acid, peak 13 hydroxysafflor yellow A, peak 16 paeoniflorin, peak 20 forsythiaside A, peak 21 ferulic acid, peak 29 berberine hydrochloride, peak 32 arctiin, peak 40 glycyrrhizic acid, peak 41 paeonol, peak 47 emodin, peak 51 emodin, peak 53 emodin methyl ether, peak 54 rubiacin. By further analyzing each negative sample, the source of each common peak was confirmed, thus achieving comprehensive and systematic effective quality detection of the Dermatosis Xuedu Pills. This quality detection method has the advantages of being simple and rapid, stable and reliable, highly precise, reproducible, and easy to master.

[0097] 4. The fingerprint spectrum and multi-component content determination method of the Skin Disease Blood Toxin Pills provided by the present invention, on the basis of the fingerprint spectrum evaluation, further determine the contents of 7 components including gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol and madderol. This method can more comprehensively and systematically reflect the quality of the Skin Disease Blood Toxin Pills than the single component content determination.

[0098] 5. The fingerprint spectrum and multi-component content determination method of the skin disease blood poison pill provided by the present invention have been verified by fingerprint spectrum detection methodology and content determination methodology, respectively, proving that the method has the advantages of strong specificity, good precision, good repeatability, good stability and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 This is the matching map for 15 batches of samples of Dermatosis Xuedu Pills;

[0100] Figure 2 The reference substances are located on the chromatogram; 1. Gallic acid, 2. Chlorogenic acid, 3. Hydroxysafflor yellow A, 4. Paeoniflorin, 5. Forsythiaside A, 6. Ferulic acid, 7. Berberine hydrochloride, 8. Arctiin, 9. Glycyrrhizic acid, 10. Paeonol, 11. Rhaponticin, 12. Rhaponticol, 13. Rhaponticin methyl ether, 14. Rubiatin;

[0101] Figure 3This is the common peak attribution diagram of negative samples; S1: Dermatological Disease Xuedu Pills full prescription powder; S2: Xanthium sibiricum, honeysuckle, and honeysuckle are negative; S3: Red peony root and white peony root are negative; S4: Rhubarb is negative; S5: Angelica sinensis and Chuanxiong are negative; S6: Licorice is negative; S7: Carthamus tinctorius is negative; S8: Phellodendron amurense is negative; S9: Forsythia suspensa is negative; S10: Paeonia suffruticosa is negative; S11: Arctium lappa is negative; S12 Rubia cordifolia is negative; S13 Prunus persica and bitter almond are negative;

[0102] Figure 4 This is the reference fingerprint of Dermatosis Xuedu Pills; 5. Gallic acid, 11. Chlorogenic acid, 13. Hydroxysafflor yellow A, 16. Paeoniflorin, 20. Forsythiaside A, 21. Ferulic acid, 29. Berberine hydrochloride, 32. Arctiin, 40. Glycyrrhizic acid, 41. Paeonol, 47. Rhamnolide, 51. Rhamnolide, 53. Rhamnolide methyl ether, 44. Rubiatin;

[0103] Figure 5 is a chromatogram of gradient program one;

[0104] Figure 6 is the gradient program dichroic chromatogram;

[0105] Figure 7 Three chromatograms for the gradient program;

[0106] Figure 8 This is the chromatogram of gradient program four;

[0107] Fig. 9 Chromatogram for gradient program V;

[0108] Fig.10 Chromatogram at 230nm\280nm with formic acid added to the mobile phase;

[0109] Fig.11 Chromatogram of 230nm\280nm for adding glacial acetic acid into the mobile phase;

[0110] Fig.12 The chromatograms are detected at wavelengths of 230 nm and 280 nm;

[0111] Fig.13 The chromatograms are for phosphoric acid dosages of 0.05%, 0.10%, and 0.15%;

[0112] Fig.14 The chromatograms are at flow rates of 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min;

[0113] Fig.15 The chromatograms are at column temperatures of 25°C, 30°C, and 35°C;

[0114] Fig.16 Chromatogram for specificity detection;

[0115] Fig.17 The chromatograms are compared between Comparative Example 1 and Example 1;

[0116] Fig.18 The chromatogram is compared between Comparative Example 4 and Example 1;

[0117] Fig.19 The chromatogram is compared between Comparative Example 5 and Example 1;

[0118] Fig. 20 This is the chromatographic peak matching diagram of the precision test (n=6);

[0119] Fig.21 This is the chromatographic peak matching diagram of the repeatability test (n=6);

[0120] Fig. 22 This is the solution stability chromatographic peak matching diagram (n=6). DETAILED DESCRIPTION

[0121] In order to make the technical means, creative features, purpose and effect of the present invention easy to understand, the present invention is further explained below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without making creative work all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited. The technology and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0122] Instruments and reagents

[0123] Agilent 1260 InfinityⅡ liquid chromatograph (vacuum degasser, binary pump, autosampler, column oven, DAD detector); analytical balance, model: Mettler Toledo PL203 and XS205; KQ-300DB digitally controlled ultrasonic cleaner (300W, 40kHz).

[0124] Phosphoric acid (AR), Lot: 20220915, Manufacturer: Sinopharm Chemical Reagent Co., Ltd.;

[0125] Methanol (AR), Lot: 20220412, Manufacturer: Sinopharm Chemical Reagent Co., Ltd.;

[0126] Acetonitrile (HPLC), Lot: SHBP1023, Manufacturer: Merck;

[0127] Water, purified water.

[0128] Sample: Dermatopathy Xuedu Pills (produced by Beijing Tongrentang Pharmaceutical Co., Ltd.).

[0129] Reference substances: gallic acid, chlorogenic acid, hydroxysafflor yellow A, paeoniflorin, forsythiaside A, ferulic acid, astilbin, forsythiaside, berberine hydrochloride, arctiin, glycyrrhizic acid, paeonol, emodin, catalpol, chrysophanol, physophanol methyl ether, madderol, and amygdalin were all purchased from China Food and Drug Inspection Institutes.

[0130] Example 1. Determination of component content

[0131] S1: Preparation of reference solution: Take appropriate amount of gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol, and maddercorus odoratus reference substances, accurately weigh them, and add methanol to prepare reference solutions containing 7.478 μg / mL of gallic acid, 20.036 μg / mL of chlorogenic acid, 30.198 μg / mL of paeoniflorin, 6.422 μg / mL of berberine hydrochloride, 8.702 μg / mL of arctiin, 4.824 μg / mL of paeonol, and 3.672 μg / mL of maddercorus odoratus per 1 mL.

[0132] S2: Preparation of test solution: Take an appropriate amount of Dermatopathy Blood Toxin Pills, grind them into powder, take about 1.0 g, weigh it accurately, put it in a stoppered conical flask, accurately add 50 mL of 80% methanol, weigh it, extract it with ultrasound (power 500 W, 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0133] S3: Accurately pipette 10μL of reference solution and 10μL of test solution, inject them into liquid chromatograph respectively, and measure to obtain the result.

[0134] The chromatographic conditions were as follows: using octadecylsilane bonded silica gel as a filler (chromatographic column: Platisil ODS 5um, 4.6×250mm); using acetonitrile solution as mobile phase A, using an aqueous solution containing 0.1% phosphoric acid as mobile phase B, gradient elution according to the provisions in Table 1, detection wavelength of 230nm, flow rate of 1mL·min -1 ; Column temperature is 30°C. The number of theoretical plates calculated based on berberine hydrochloride should not be less than 5000.

[0135] Table 1. Elution gradient

[0136] Time (min) Mobile phase A (%) Mobile phase B (%) 0 2 98 8 10 90 15 14 86 20 16 84 35 23 77 50 40 60 60 70 30 70 80 20 80 90 10

[0137] The above method was used to analyze 15 batches of skin disease blood poison pill samples produced from 2021 to 2023.

[0138] like Figure 1The content of each component at 230 nm was calculated as shown in Table 2. The content determination results of 15 batches of test products are shown in Table 2 below.

[0139] Table 2. Content determination results of the 1st to 8th batches of Dermatosis Blood Poison Pills mg / g

[0140]

[0141]

[0142] Table 3. Content determination results of the 9th to 15th batches of Dermatosis Blood Poison Pills mg / g

[0143]

[0144] Example 2. Fingerprint analysis

[0145] S1: prepare 0.1 mg / mL reference solution of gallic acid, chlorogenic acid, hydroxysafflor yellow A, paeoniflorin, forsythiaside A, ferulic acid, berberine hydrochloride, arctiin, glycyrrhizic acid, paeonol, emodin, chrysophanol, physophanol methyl ether, and rubiacorin reference substances with methanol;

[0146] S2: Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0g, accurately weigh it, put it in a stoppered conical flask, accurately add 50mL of 80% methanol, weigh it, extract it by ultrasound (power 500W, 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0147] S3: Preparation of negative test solution: Prepare the test sample lacking madder according to the prescription, grind into powder, pass through No. 3 sieve, take about 1.0g of fine powder, accurately weigh, and prepare madder negative test solution according to the test solution preparation method. Prepare negative test solutions of red peony root and white peony root, angelica and ligusticum, xanthium, honeysuckle and honeysuckle vine, forsythia, phellodendron, bitter almond and peach kernel, arctium, peony bark, rhubarb, licorice, and safflower in the same way;

[0148] S4: respectively aspirate the reference solution, the test solution, and the negative test solution, and inject them into a liquid chromatograph, respectively, and measure to obtain a chromatogram; the chromatographic conditions are the same as those in Example 1.

[0149] The chromatogram of the test solution has a chromatographic peak with the same retention time as the single reference substance in the chromatogram of the reference solution, and the spectrum of the corresponding chromatographic peak Figure 1 This allows the compound names of the various peaks in the chromatogram of the test product to be confirmed.

[0150] like Figure 2As shown, 1. Gallic acid, 2. Chlorogenic acid, 3. Hydroxysafflor yellow A, 4. Paeoniflorin, 5. Forsythiaside A, 6. Ferulic acid, 7. Berberine hydrochloride, 8. Arctiin, 9. Glycyrrhizic acid, 10. Paeonol, 11. Rhaponticol, 12. Rhaponticol, 13. Rhaponticol methyl ether, 14. Rubiatin.

[0151] Table 4. Retention time (RT) of the corresponding peaks in the chromatograms of the reference and test products

[0152]

[0153] According to the chromatograms of each negative sample and the test solution ( Figure 3 ), analyze the attribution of each common peak, among which peaks 5, 28, 33, 34, 35, 38, 43, 44, 47, 48, 51, 53 are from rhubarb; peaks 7, 20, 31 are from forsythia; peaks 8, 11, 27 are from xanthium sibiricum, honeysuckle and honeysuckle; peaks 12 and 40 are from liquorice; peaks 15 and 16 are from red peony root and white peony root; peaks 21 and 49 are from angelica and ligusticum; peak 29 is from phellodendron; peaks 32 and 42 are from arctium fruit; peaks 37 and 54 are from madder; peak 41 is from moutan bark; other common peaks may come from other medicinal pieces, and cannot be assigned to them due to their low content.

[0154] According to the above method, 15 batches of Dermatosis Blood Poison Pill samples produced from 2021 to 2023 were analyzed. The chromatograms of the 15 batches of Dermatosis Blood Poison Pill were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", multi-point correction, full peak matching, and 54 chromatographic peaks with a matching number of 15, that is, 54 common peaks. The Dermatosis Blood Poison Pill control fingerprint was generated from the fingerprints of the 15 batches of test samples.

[0155] The reference substance was used as a reference to calibrate 14 known compounds, and the 29th peak berberine hydrochloride was used as the reference peak (S). The relative retention time of berberine hydrochloride was 1.00, and the relative retention time of other peaks was calculated. The relative retention time of each compound was as shown in Table 5 below:

[0156] Table 5. Relative retention times

[0157] name Relative retention time gallic acid 0.23 Chlorogenic acid 0.45 Hydroxysafflor Yellow A 0.49 Paeoniflorin 0.61 Forsythiaside A 0.75 Ferulic acid 0.77 Berberine Hydrochloride 1.00 Arctiin 1.06 Glycyrrhizic acid 1.28 Paeonol 1.30 Rhein 1.48 Chrysophanol 1.61 Physcion 1.67 Rubiatin 1.75

[0158] like Figure 4 As shown, the peaks are:

[0159] 5. Gallic acid, 11. Chlorogenic acid, 13. Hydroxysafflor yellow A, 16. Paeoniflorin, 20. Forsythiaside A, 21. Ferulic acid, 29. Berberine hydrochloride, 32. Arctiin, 40. Glycyrrhizic acid, 41. Paeonol, 47. Rhamnolide, 51. Rhamnolide, 53. Rhamnolide methyl ether, 54. Rubiatin.

[0160] Example 3. Selection of mobile phase gradient elution program

[0161] Chromatographic column: Platisil ODS 5um, 4.6×250mm; detection wavelength: 190nm-400nm; acetonitrile (A)-0.1% phosphoric acid (B) as mobile phase, flow rate 1mL·min -1 ; The column temperature was 30°C. The gradient elution program was determined through multiple experiments.

[0162] Reference solution: Single reference solution of gallic acid, chlorogenic acid, hydroxysafflor yellow A, paeoniflorin, forsythiaside A, ferulic acid, berberine hydrochloride, forsythiaside, arctiin, glycyrrhizic acid, paeonol, emodin, catalpol, chrysophanol, physophanol methyl ether, and rubiacin are used to locate each active ingredient on the chromatogram.

[0163] Test solution: Take an appropriate amount of Dermatopathy Blood Toxin Pills, grind them into powder, take about 1 g, weigh accurately, put it in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper it tightly, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0164] Determination method: Accurately pipette 10μL of reference solution and 10μL of test solution, inject them into liquid chromatograph respectively, and determine.

[0165] a. Gradient Program 1

[0166] Table 6.

[0167] time Mobile phase Mobile phase B 0 3 97 15 15 85 25 20 80 45 48 52 52 80 20 60 80 20 75 100 0 80 100 0

[0168] b. Gradient Program 2

[0169] Table 7.

[0170]

[0171]

[0172] c. Gradient Program 3

[0173] Table 8.

[0174] time Mobile phase Mobile phase B 0 2 98 8 10 90 35 25 75 50 40 60 60 70 30 70 80 20 80 100 0

[0175] d. Gradient Program 4

[0176] Table 9.

[0177] time Mobile phase Mobile phase B 0 2 98 8 10 90 35 25 75 40 30 70 50 58 42 65 80 20 70 75 25 80 100 0

[0178] e. Gradient Program 5

[0179] Table 10.

[0180] time Mobile phase Mobile phase B 0 2 98 8 10 90 15 14 86 20 16 84 35 23 77 50 40 60 60 70 30 70 80 20 80 90 10

[0181] The results show that: Figure 5-9 As shown, the chromatogram obtained by gradient program 1 ( Figure 5 ), there is accumulation of chromatographic peaks at retention time 20-50min, and the chromatographic peaks have not reached baseline separation; the chromatogram obtained by gradient program 2 ( Figure 6 ), there is accumulation of chromatographic peaks at retention time 30-50min, and the chromatographic peaks have not reached baseline separation; the chromatogram obtained by gradient program three ( Figure 7 ), there is accumulation of chromatographic peaks between retention time 35-45min, and the chromatographic peaks have not reached baseline separation; the chromatogram obtained by gradient program 4 ( Figure 8 ), there is accumulation of chromatographic peaks between retention time 40-45min, and the chromatographic peaks are not separated by baseline; the chromatogram obtained by gradient program 5 ( Fig. 9 ), the baseline is stable and the chromatographic peaks have good separation.

[0182] Example 4. Selection of acidic additives in mobile phase

[0183] a. Formic acid

[0184] The mobile phase A was acetonitrile, and the mobile phase B was 0.1% formic acid. Gradient elution was performed according to gradient program five.

[0185] Results: See the results Fig.10 , formic acid was added to the mobile phase. Under the condition of 280nm, the chromatographic peaks were well separated and the baseline was stable; under the condition of 230nm, the baseline fluctuated greatly.

[0186] b. Glacial acetic acid

[0187] The mobile phase A was acetonitrile, and the mobile phase B was 0.2% glacial acetic acid. Gradient elution was performed according to gradient program five.

[0188] Results: See the results Fig.11 , formic acid was added to the mobile phase. Under the condition of 280nm, the chromatographic peaks were well separated and the baseline was stable; under the condition of 230nm, the baseline fluctuated greatly.

[0189] Example 5. Selection of detection wavelength

[0190] The samples were detected at full wavelength using a diode array detector. According to the collected three-dimensional spectra, more chromatographic peaks were detected at wavelengths of 210-290 nm. The chromatograms obtained at wavelengths of 230 nm and 280 nm ( Fig.12) It can be seen that more chromatographic peaks can be detected at 230nm, and 230nm is preferably used as the detection wavelength for fingerprint and multi-component content determination.

[0191] In Example 3-5, acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase, and the gradient elution program was optimized through the optimization of the gradient elution program; 0.1% formic acid, 0.2% glacial acetic acid aqueous solution and nitrile were eluted according to the preferred gradient program, and the results showed that the baseline fluctuated greatly at the preferred detection wavelength of 230nm; 0.1% phosphoric acid-acetonitrile aqueous solution was used as the mobile phase, and there was a good baseline in the wavelength region of 210-400nm, and the skin disease blood poison pills contained complex ingredients. According to the chromatogram, more component peaks were measured at a lower wavelength of 230nm. Therefore, 0.1% phosphoric acid-acetonitrile aqueous solution was preferred as the mobile phase, and the wavelength was preferably 230nm.

[0192] Example 6. Screening of chromatographic columns

[0193] Acetonitrile-0.1% phosphoric acid aqueous solution was used as the mobile phase, and gradient elution was performed according to gradient program 5 at a flow rate of 1 mL min -1 ; Column temperature is 30℃; Detection wavelength is 230nm. The effects of three brands of chromatographic columns on detection are compared, and the test results are shown in Table 11.

[0194] Table 11. Different chromatographic columns and test results

[0195]

[0196] Conclusion: All three chromatographic columns can be used for the determination of this product, and the preferred chromatographic column is Platisil ODS 5um, 4.6×250mm.

[0197] Example 7. Screening of the amount of phosphoric acid in the mobile phase

[0198] The other conditions were the same as those in Example 1, and the effects of 0.05%, 0.10% and 0.15% phosphoric acid in the phosphoric acid aqueous solution in the mobile phase on the detection results of the same sample were compared.

[0199] The results show that when the amount of phosphoric acid in the phosphoric acid aqueous solution is 0.05%, 0.10% and 0.15%, the chromatographic Fig.13 There is no significant difference in the peak shape and separation degree of each peak. Therefore, the amount of phosphoric acid in the mobile phase between 0.05% and 0.15% meets the detection requirements, and the preferred amount of phosphoric acid in the mobile phase is 0.10%.

[0200] Example 8. Screening of flow rate

[0201] The other conditions were the same as those in Example 1, and the effects of flow rates of 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min on the test results of the same sample were compared.

[0202] The results showed that when the flow rates were set to 0.9 mL / min, 1.0 mL / min and 1.1 mL / min, the chromatographic Fig.14 It can be seen that there is no significant difference in the test results for the same sample. Therefore, the flow rate between 0.9mL / min-1.1mL / min is sufficient for the detection of this product, and the preferred mobile phase flow rate is 1.0mL / min.

[0203] Example 9. Screening of column temperature

[0204] Other conditions were the same as those in Example 1, and the effects of column temperatures of 25°C, 30°C, and 35°C on the test results of the same sample were compared.

[0205] The results showed that when the column temperature was set at 25℃, 30℃ and 35℃, the chromatographic Fig.15 It can be seen that there is no significant difference in the test results for the same sample. Therefore, a column temperature of 30°C-35°C can meet the test of this product, and the preferred column temperature is 30°C.

[0206] Example 10. Selection of extraction solvent

[0207] Compared with Example 1, only the volume fraction of methanol in S2 was changed, and 50 mL of 60%, 70%, 80%, and 100% methanol were accurately added, respectively, the chromatograms were recorded, and the contents of the seven components in the test solution were calculated by the area external standard method.

[0208] The result data are shown in Table 12. 70% or 80% methanol solution as solvent has a higher total content of 7 components in Skin Disease Blood Toxin Pills. Using 80% methanol as extraction solvent has less interference from impurity peaks, so 80% methanol is preferred as extraction solvent.

[0209] Table 12. Comparative content results of the extraction solvents of the test products

[0210] Extraction solvent 60% methanol 70% methanol 80% Methanol 100% Methanol Gallic acid (mg / g) 0.3576 0.3581 0.3479 0.2347 Chlorogenic acid (mg / g) 1.2197 1.2564 1.1831 0.8257 Paeoniflorin (mg / g) 1.5514 1.5413 1.5578 1.3772 Berberine hydrochloride (mg / g) 0.2328 0.3265 0.3991 0.3845 Arctiin (mg / g) 0.4705 0.4704 0.4751 0.4530 Paeonol(mg / g) 0.3069 0.3138 0.3156 0.3064 Rubiatin(mg / g) 0.1336 0.2451 0.2141 0.2350 Total (mg / g) 4.2724 4.5117 4.4926 3.8165

[0211] Example 11. Selection of extraction method and extraction time

[0212] Take the same batch of Skin Disease Blood Toxin Pills, and compare with Example 1, only change the extraction time, ultrasonic extraction (power 500 W, 40 kHz) for 30, 45, and 60 minutes respectively, take out, let cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and obtain.

[0213] The content results are shown in Table 13 below. The total content of each component is higher after ultrasonic extraction for 30 minutes, so ultrasonic extraction for 30 minutes is preferred for the preparation of the test solution.

[0214] Table 13. Comparison of extraction method and extraction time Content results (mg / g)

[0215]

[0216]

[0217] Example 12. Specificity of the method

[0218] According to the preferred fingerprint and multi-component content determination method, accurately measure 10 μL of blank solvent (80% methanol) and test solution, inject them into liquid chromatograph respectively, and determine them to obtain the result.

[0219] Results: Fig.16 As shown, the blank solvent (80% methanol) had no interference with the detection of the test product.

[0220] Comparative Example 1

[0221] Compared with Example 1, the mobile phase was methanol-0.1% phosphoric acid aqueous solution (22:78),

[0222] The other operations are the same as those in Example 1.

[0223] Results: See Fig.17 It is obvious that when the Dermatosis Blood Poison Pill sample was analyzed by isocratic elution of the mobile phase, only four chromatographic peaks were separated, and the front peak was fast, resulting in chromatographic peak accumulation, and most of the components were retained in the chromatographic column and not eluted. Example 1 has a better separation effect on the same sample.

[0224] Comparative Example 2

[0225] Compared with Example 1,

[0226] S2: Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0g, accurately weigh it, put it in a stoppered conical flask, accurately add 50mL of 80% ethanol, weigh it, extract it by ultrasound (power 500W, 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 80% ethanol, shake it well, filter it, and take the filtrate to obtain it.

[0227] The other operations are the same as those in Example 1.

[0228] Results: With 80% ethanol as the extraction solvent, the average content of gallic acid and chlorogenic acid was lower than that in Example 1 and the relative standard deviation of the repeatability of the method was larger, indicating that the extraction effect of the present invention with 80% methanol as the solvent is better than that with 80% ethanol as the solvent.

[0229] Comparative Example 3

[0230] Compared with Example 1,

[0231] S2: Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 1.0g, accurately weigh it, put it in a stoppered conical flask, accurately add 50mL of 80% acetonitrile, weigh it, extract it by ultrasound (power 500W, 40kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 80% acetonitrile, shake it well, filter it, and take the filtrate to obtain it.

[0232] The other operations are the same as those in Example 1.

[0233] Results: With 80% acetonitrile as the extraction solvent, the average contents of gallic acid, chlorogenic acid and paeoniflorin were lower than those in Example 1 and the relative standard deviation of the repeatability of the method was larger, indicating that the extraction effect of the present invention with 80% methanol as the solvent is better than that with 80% acetonitrile as the solvent.

[0234] Comparative Example 4

[0235] Compared with Example 1, the mobile phase elution gradient of the reference document "HPLC fingerprint study of skin disease blood poison pills" is:

[0236] 0-8.5 min, 3%-8% mobile phase A, 97%-92% mobile phase B;

[0237] 8.5-13 min, 8%-15% mobile phase A, 92%-85% mobile phase B;

[0238] 13-30 min, 15%-26% mobile phase A, 85%-74% mobile phase B;

[0239] 30-31 min, 26%-29% mobile phase A, 74%-71% mobile phase B;

[0240] 31-40 min, 29%-48% mobile phase A, 71%-52% mobile phase B;

[0241] 40-50 min, 48%-80% mobile phase A, 52%-20% mobile phase B;

[0242] 50-55 min, 80%-95% mobile phase A, 20%-5% mobile phase B;

[0243] 55-60 min, 95%-100% mobile phase A, 5%-0% mobile phase B;

[0244] The other operations are the same as those in Example 1.

[0245] Results: The mobile phase gradient program of Comparative Example 4 was used to analyze the skin disease blood poison pill sample. The measured results are shown in Fig.18Compared with Example 1, it can be seen that the paeonol peak in the chromatogram of Comparative Example 4 is not completely separated from the adjacent peaks, and the baseline moves up between the retention time of 55-65min, and the separation effect of chrysophanol, chrysophanol and physophanol methyl ether is not good.

[0246] The above results were produced because the gradient program of Comparative Example 4 was between 40 and 60 min, and the proportion of acetonitrile in mobile phase A was too large, resulting in poor separation of multiple components and unstable baseline.

[0247] Comparative Example 5

[0248] Compared with Example 1, the elution gradient of the reference document "HPLC fingerprint study of skin disease blood poison tablets and determination of the contents of three components" is:

[0249] 0-20min, 3%-15% mobile phase A, 97%-85% mobile phase B;

[0250] 20-40 min, 15%-25% mobile phase A, 85%-75% mobile phase B;

[0251] 40-45 min, 25%-30% mobile phase A, 75%-70% mobile phase B;

[0252] 45-70 min, 30%-60% mobile phase A, 70%-40% mobile phase B;

[0253] The other operations are the same as those in Example 1.

[0254] Results: The mobile phase gradient program of Comparative Example 5 was used to analyze the skin disease blood poison pill sample. The measured results are shown in Fig.19 Compared with Example 1, there are multiple chromatographic peaks that are not eluted and separated between the retention time of 60-70 min.

[0255] The above results were produced because the mobile phase gradient program of Comparative Example 5 ended elution at 70 min when the maximum proportion of acetonitrile in mobile phase A reached 60%, resulting in failure to separate multiple components such as chrysophanol, physophanol methyl ether and rubiacein.

[0256] Test example

[0257] 1. Precision test

[0258] Take a sample of Dermatosis Xuedu Pills (batch number 22082145) and prepare a test solution according to Example 1. Take 10 μL of the same test solution, inject it 6 times in succession, and record the chromatogram.

[0259] Results: Fig. 20As shown, six chromatograms obtained by continuous injection of the same test solution were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine", with the first injection chromatogram (S1) as a reference, a time width of 0.1, multi-point correction using the median method, full peak matching, and a matching number of 6. The similarities of the six chromatograms were all greater than 0.990, indicating that the instrument had good precision.

[0260] 2. Repeatability test

[0261] Take the same batch of Dermatosis Blood Poison Pills samples (batch number 22082145), prepare 6 test solutions in parallel according to Example 1, take the above 6 test solutions and reference solution, inject 10 μL respectively, and record the chromatogram.

[0262] Results: Fig.21 As shown, 6 test solutions from the same batch of samples were prepared by the same method. The 6 chromatograms obtained by injection were respectively introduced into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine", with the first sample chromatogram (S1) as a reference, the time width was 0.1, the median method was used for multi-point correction, full peak matching, the number of matches was 6, and the similarity of the 6 chromatograms was greater than 0.990, indicating that the method has good repeatability.

[0263] 3. Test solution stability test

[0264] Take a sample of Dermatosis Xuedu Pills (batch number 22082145) and prepare a test solution according to Example 1. The newly prepared test solution is placed at room temperature, and samples are injected at 0, 4, 8, 12, 16, and 24 hours, and the chromatograms are recorded.

[0265] Results: The results of Example 1 are as follows Fig. 22 As shown, the chromatograms of the same test solution measured at different time points Figure 6 The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" was imported, the chromatogram measured at the 0th hour (S1) was used as a reference, the time width was 0.1, multi-point correction was performed using the median method, all peaks were matched, the number of matches was 6, and the similarities of the 6 chromatograms were all greater than 0.900, indicating that the solution had good stability for 24 hours under room temperature conditions.

[0266] 4. Validation of multi-component content determination methodology

[0267] 4.1 Linear relationship and range

[0268] Take appropriate amount of gallic acid, chlorogenic acid, berberine hydrochloride, arctiin, paeonol, and marigoldin reference substances, accurately weigh them, and dilute them with 80% methanol to make a reference substance mixed solution with a certain concentration gradient. Take 10μL of the reference substance mixed solution from low to high concentration, respectively, and measure to examine the linear relationship between the concentration (μg / mL) of each component and the corresponding peak area. Results Gallic acid, chlorogenic acid, berberine hydrochloride, arctiin, paeonol, and marigoldin reference substances have a good linear relationship with the corresponding area, and the linear range of the injection amount of each component meets the detection requirements.

[0269] Table 14. Results of investigation on linear relationship of each component

[0270]

[0271]

[0272] 4.2 Repeatability test

[0273] Take the chromatogram obtained under the "Repeatability Test" item of fingerprint method validation ( Fig.21 ), calculate the average content and relative standard deviation of Example 1 and Comparative Examples 2-3. The results are shown in Tables 15 and 16. The RSD values ​​of each compound in the 6 parallel test samples measured in Example 1 are all ≤3.0%, indicating that the repeatability of the method is good; the RSD values ​​of each compound in the 6 parallel test samples measured in Comparative Examples 2-3 are all ≤4.0%. The average content of gallic acid and chlorogenic acid measured in Comparative Example 2 is lower than that in Example 1; the average content of gallic acid, chlorogenic acid and paeoniflorin measured in Comparative Example 3 is lower than that in Example 1.

[0274] Conclusion: The content determination results of 7 compounds and the repeatability of the method in Example 1 are better than those in Comparative Examples 2 and 3.

[0275] Table 15. Repeatability test results of Example 1 (n=6)

[0276]

[0277] Table 16. Average values ​​and RSD values ​​of each component of Example 1 and Comparative Examples 2-3 (n=6)

[0278]

[0279]

[0280] 4.3 Accuracy test

[0281] Preparation of the test solution: Take an appropriate amount of the known content of the skin disease blood poison pill sample (batch number 22082145), take half of the standard sample amount of the test sample, accurately weigh, respectively put in a stoppered conical bottle, add an appropriate amount of the reference substance, parallel 6 parts, according to the test solution preparation method of Example 1 and Comparative Examples 2-3, make a test solution. Accurately draw 10 μL of the reference solution and the test solution, respectively inject them into the liquid chromatograph, measure, calculate the sample recovery rate, and the average recovery rate of the 7 compounds is between 98% and 101%, and the RSD value of the recovery rate of each compound measured in 6 samples is ≤2.19%, which is significantly better than Comparative Examples 2-3, and meets the requirements for the sample recovery rate determination, indicating that the accuracy of the method is good.

[0282] Table 17. Accuracy test results of Example 1 (n=6)

[0283]

[0284] Table 18. Accuracy test results of Example 1 and Comparative Examples 2-3 (n=6)

[0285]

[0286]

[0287] 4.4 Stability of reference solution

[0288] Take a newly diluted reference solution, place it at room temperature, inject samples at different time points, measure the samples according to Example 1, and record the chromatogram. The results show that the RSD values ​​of each peak area measured by the reference solution at room temperature for 25 hours are ≤3.0%, which proves that the reference solution has good stability when placed at room temperature for 25 hours.

[0289] Table 19. Results of stability test of reference solution (n=6)

[0290]

[0291] The present invention compares the influence of different column temperatures, flow rates, phosphoric acid dosage in the mobile phase and slight changes in the mobile phase gradient program on the detection of the fingerprint spectrum and multi-component content of the skin disease blood poison pill. The results show that under the premise of the preferred chromatographic column PlatisilODS 5um, 4.6×250mm, the slight changes in the column temperature of 30℃-35℃, the flow rate of 1.0mL / min-1.1mL / min and the addition of phosphoric acid in the mobile phase from 0.05%-0.15% have no obvious effect on the detection results, and the method has good durability.

[0292] The present invention carries out methodological verification for fingerprint detection and multi-component content determination, and the results of methodological verification show that the specificity, repeatability, stability of the solution, etc. of the method meet the requirements of fingerprint detection; the results of methodological verification for multi-component content determination show that the linearity and linear range of the method, the repeatability of the method, the recovery rate, the stability of the solution, the durability of the method, etc. meet the detection requirements of multi-component content determination.

[0293] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining the content of components of Dermatopathy Xuedu Pills, characterized in that: The ingredients include gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol and rubiacein, and include the following steps: S1: Prepare reference solution: weigh reference substances gallic acid, chlorogenic acid, paeoniflorin, berberine hydrochloride, arctiin, paeonol, and rubiacein, and mix them with solvent to prepare a mixed reference solution; S2: Prepare the test solution: weigh the skin disease blood poison pills, mix them with the solvent, weigh them, extract, cool, weigh them again, make up the lost weight with the solvent, shake well, filter, and take the filtrate; the solvent is a methanol aqueous solution; the volume fraction of methanol in the methanol aqueous solution is 80%; S3: Pipette the reference solution and the test solution, inject them into the liquid chromatograph respectively, measure and obtain the chromatogram; Column: Platisil ODS, 5um, 4.6×250mm; Mobile phase: acetonitrile solution is mobile phase A, and 0.1% volume fraction of phosphoric acid aqueous solution is mobile phase B; Detection wavelength: 230nm; Flow rate: 1 mL min -1 ; Column temperature: 30°C; The elution method is gradient elution, and the elution gradient is: 0-8min, 2%-10% mobile phase A, 98%-90% mobile phase B; 8-15 min, 10%-14% mobile phase A, 90%-86% mobile phase B; 15-20 min, 14%-16% mobile phase A, 86%-84% mobile phase B; 20-35 min, 16%-23% mobile phase A, 84%-77% mobile phase B; 35-50 min, 23%-40% mobile phase A, 77%-60% mobile phase B; 50-60 min, 40%-70% mobile phase A, 60%-30% mobile phase B; 60-70 min, 70%-80% mobile phase A, 30%-20% mobile phase B; 70-80min, 80%-90% mobile phase A, 20%-10% mobile phase B.

2. The measuring method according to claim 1, characterized in that The concentration of each reference substance in the mixed reference substance solution described in S1 is: gallic acid 7-9 μg / mL, chlorogenic acid 18-22 μg / mL, paeoniflorin 28-32 μg / mL, berberine hydrochloride 5-7 μg / mL, arctiin 8-10 μg / mL, paeonol 4-6 μg / mL, and madderin 3-5 μg / mL.

3. The measuring method according to claim 1, characterized in that The time of S2 ultrasonic extraction is 30-60min, the power of ultrasound is 450-600W, and the frequency is 35-45kHz.

4. A method for constructing a fingerprint spectrum, characterized in that: The following steps are involved: S1: Prepare reference substance solution: weigh reference substances such as gallic acid, chlorogenic acid, hydroxysafflor yellow A, paeoniflorin, forsythiaside A, ferulic acid, berberine hydrochloride, arctiin, ammonium glycyrrhizinate, paeonol, emodin, chrysophanol, physophanol methyl ether, and rubia cordifolia, respectively, and mix them with solvent to obtain reference substance solution; S2: Prepare the test solution: weigh the sample, mix it with the solvent, extract it, cool it, weigh it, make up the lost weight with the solvent, shake it well, filter it, and take the filtrate to obtain the solution; the solvent is a methanol aqueous solution; the volume fraction of methanol in the methanol aqueous solution is 80%; S3: preparing negative test solution: respectively weigh test samples lacking madder, lacking red peony root and white peony root, lacking angelica and ligusticum, lacking Xanthium sibiricum, honeysuckle and honeysuckle, lacking Forsythia suspensa, lacking Phellodendron amurense, lacking Apricot kernel and peach kernel, lacking Arctium lappa, lacking Paeonia suffruticosa, lacking Rhubarb, lacking Licorice, lacking Carthamus tinctorius, and prepare negative test solutions according to the steps of S2; S4: respectively aspirate the reference solution, the test solution, and the negative test solution, and inject them into the liquid chromatograph, respectively, and measure to obtain a chromatogram; The chromatographic conditions are: Column: Platisil ODS, 5um, 4.6×250mm; Mobile phase: acetonitrile solution is mobile phase A, and 0.1% volume fraction of phosphoric acid aqueous solution is mobile phase B; Detection wavelength: 230nm; Flow rate: 1 mL min -1 ; Column temperature: 30°C; The elution method is gradient elution, and the elution gradient is: 0-8min, 2%-10% mobile phase A, 98%-90% mobile phase B; 8-15 min, 10%-14% mobile phase A, 90%-86% mobile phase B; 15-20 min, 14%-16% mobile phase A, 86%-84% mobile phase B; 20-35 min, 16%-23% mobile phase A, 84%-77% mobile phase B; 35-50 min, 23%-40% mobile phase A, 77%-60% mobile phase B; 50-60 min, 40%-70% mobile phase A, 60%-30% mobile phase B; 60-70 min, 70%-80% mobile phase A, 30%-20% mobile phase B; 70-80min, 80%-90% mobile phase A, 20%-10% mobile phase B.

5. The construction method according to claim 4, characterized in that: The concentration of the reference solution described in S1 is 0.08-0.12 mg / mL.

6. The construction method according to claim 4, characterized in that: The time of S2 ultrasonic extraction is 30-60min, the power of ultrasound is 450-600W, and the frequency is 35-45kHz.

7. Application of the method for determining the component content of the Dermatosis Blood Toxin Pills according to any one of claims 1 to 3 or the method for constructing the fingerprint spectrum according to any one of claims 4 to 6 in the quality control of Dermatosis Blood Toxin Pills preparations.

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