Fingerprint spectrum detection method of consistent decoction reference sample and application of fingerprint spectrum detection method

Abstract

The invention discloses a fingerprint spectrum detection method of a consistent decoction reference sample and application, and relates to the technical field of traditional Chinese medicine detection. According to the fingerprint detection method, a test solution is determined and analyzed by adopting a high performance liquid chromatography, and the conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 chromatographic column; the column temperature is 28-32 DEG C; the detection wavelength is 0 to 25 minutes, and the wavelength is 278 to 282nm; in 25-66 minutes, the wavelength is 318-322 nm; the time is 66 to 80 minutes, and the wavelength is 278 to 282nm; the time is 80 to 90 minutes, and the wavelength is 318 to 322 nm; the mobile phase A is acetonitrile, and the mobile phase B is a 0.09-0.11 wt% phosphoric acid solution; and eluting by adopting a gradient elution procedure. According to the method, digitonin C, 4-cumaric acid, ferulic acid, rutin, verbascoside, senkyunolide I and ligustilide in the consistent decoction can be detected at a time, and the method is high in accuracy, good in repeatability, high in durability and good in specificity.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing technology, specifically to a fingerprint spectrum detection method and application for a standard sample of traditional Chinese medicine decoction. Background Technology

[0002] The Yi Guan Jian formula originates from *Yi Fang Jie Du* written by Qian Minjie in the Qing Dynasty. It consists of six ingredients: North American ginseng, Ophiopogon japonicus, Angelica sinensis, Lycium barbarum, Rehmannia glutinosa, and Melia toosendan. Decocted in water and taken orally, it nourishes Yin and soothes the liver, primarily treating liver and kidney Yin deficiency and liver Qi stagnation, such as chest, epigastric, and hypochondriac pain, acid reflux, bitter vomiting, dry throat and mouth, red tongue with little saliva, and a weak or thready pulse. It also treats hernia and abdominal masses. Modern clinical applications mainly include the treatment of gastric ulcers, gastritis, chronic hepatitis, intercostal neuralgia, hypertension, and neurosis.

[0003] The traditional Chinese medicine compound Yi Guan Jian is composed of multiple herbs, including North American ginseng, Ophiopogon japonicus, Angelica sinensis, Lycium barbarum, Rehmannia glutinosa, and Melia toosendan, all of which play important roles in the formula. However, using only one or two active ingredients like Angelica sinensis or Rehmannia glutinosa to describe the intrinsic quality of Yi Guan Jian is somewhat one-sided and directly affects its therapeutic effect. To control the efficacy of Yi Guan Jian, it is insufficient to characterize and control only one or two chemical components; the entire compound must be controlled. Traditional Chinese medicine fingerprinting plays a crucial role in modern and future quality control of traditional Chinese medicine, aligning with the holistic and fuzzy nature of traditional Chinese medicine. It can analyze the types and content distribution of effective and ineffective components in traditional Chinese medicine. With the increasing application of various technologies in the research of traditional Chinese medicine fingerprinting, it will undoubtedly play an even more important role in the quality control, research on the active components, and the study of the mechanisms of action of traditional Chinese medicine.

[0004] Chinese invention patent application CN118641659A discloses a method for detecting the content of multiple components in the traditional Chinese medicine compound Yi Guan Jian, but it uses ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry (TQLC). S MS / MS is used as an analytical tool. However, due to its high cost and limited availability in conventional production environments, its application in actual production remains somewhat limited.

[0005] Chinese invention patent application CN117007726A discloses a fingerprinting method for a traditional Chinese medicine formula, but it only identified six identifiable characteristic peaks. The chromatographic peaks identified by the reference standards were only ferulic acid, rutin, verbascoside, and ligustrol I. The number of components identified and detected simultaneously is limited, insufficient for the simultaneous detection of more active ingredients.

[0006] Chinese invention patent application CN114609291A discloses a fingerprint chromatographic detection method for Yiguanjian (a traditional Chinese medicine decoction), but it uses ultra-high performance liquid chromatography (UPLC) as the analytical method, with a flow rate of 0.25 mL / min and a duration of 90 min. This method identified 15 identifiable characteristic peaks, including chlorogenic acid, ferulic acid, verbascoside, azadirachtin, ligustilide, pyrethroid phenylethanol glycoside A1, ligustilide I, ophiopogon methylflavanone A, and ophiopogon methylflavanone B, as indicated by reference standards. This method exhibits good resolution and reproducibility under laboratory conditions. However, the UPLC system requires a pressure resistance ≥1000 bar, and the column cost is approximately 2-3 times that of a conventional HPLC column, with relatively low adoption rates among manufacturers of processed medicinal materials and traditional Chinese medicine preparations. Furthermore, the low flow rate of 0.25 mL / min cannot be directly applied to conventional HPLC equipment; scaling up the method for routine GMP batch testing requires re-validation of system suitability and robustness, significantly increasing time and economic costs.

[0007] Chinese patent application CN109406672A discloses a method for determining the fingerprint spectrum of the traditional Chinese medicine Yi Guan Jian using HPLC. The elution time of this method is 150 min, which is too long, resulting in late peak elution, large consumption of acetonitrile, and high cost. Moreover, this method cannot detect the characteristic peaks of the medicinal materials Bei Sha Shen, Mai Dong, and Chuan Lian Zi contained in Yi Guan Jian, and the information reflected is incomplete, making it difficult to effectively control the overall quality of the contained substances.

[0008] It can be seen that existing technical methods are limited by high-end instruments (such as ultra-high pressure liquid chromatography or liquid chromatography-mass spectrometry equipment), resulting in high costs and poor universality; while conventional high performance liquid chromatography detection methods can often only detect one or a few components with high accuracy, with insufficient number of identifiable characteristic peaks and low coverage of characteristic components, making it difficult to achieve rapid, economical, and simultaneous quality control of multiple indicators on conventional liquid chromatography platforms.

[0009] Therefore, it is still necessary to establish a consistent quality control method that can be implemented on a conventional HPLC platform, has wide robustness, reasonable time and economic costs, and can simultaneously detect multiple key indicator components, in order to meet the needs of rapid testing and multi-indicator detection in the production field. Summary of the Invention

[0010] This invention provides a fingerprint chromatographic detection method for a reference sample of Yiguanjian (a traditional Chinese medicine decoction). Under specific chromatographic conditions, it can efficiently and accurately detect digitalisin C, 4-coumaric acid, ferulic acid, rutin, verbascoside, ligustilide I, and ligustilide in Yiguanjian in a single step. This method overcomes the shortcomings of existing technologies. Furthermore, the method is simple to operate, rapid, accurate, repeatable, robust, and has accurate peak time. It also exhibits good specificity for the detection of each component.

[0011] This invention provides a method for detecting fingerprint spectra of a consistent reference sample.

[0012] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On the one hand, this invention provides a method for detecting fingerprint spectra of a standard sample, wherein the test solution is analyzed by high-performance liquid chromatography (HPLC), and the HPLC conditions are as follows: Chromatographic column: C18 column; Column temperature: 28℃-32℃; Detection wavelengths: 0-25 min, 278-282 nm; 25-66 min, 318-322 nm; 66-80 min, 278-282 nm; 80-90 min, 318-322 nm; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a 0.09-0.11 wt% phosphoric acid solution; Gradient elution, the elution procedure is as follows: .

[0013] The technical effects of this invention can be achieved at any point or sub-range of column temperature within the range of 28℃-32℃, including but not limited to 28℃, 29℃, 30℃, 31℃, and 32℃.

[0014] The detection wavelength is within the range of "0-25 min, 278-282 nm; 25-66 min, 318-322 nm; 66-80 min, 278-282 nm; 80-90 min, 318-322 nm". Selecting any point value or any sub-range value of the wavelength can achieve the technical effect of this invention, including but not limited to: 0-25min, 278nm; 25-66min, 318nm; 66-80min, 278nm; 80-90min, 318nm; 0-25min, 282nm; 25-66min, 322nm; 66-80min, 282nm; 80-90min, 322nm; 0-25min, 280nm; 25-66min, 320nm; 66-80min, 280nm; 80-90min, 320nm; 0-25min, 278nm; 25-66min, 322nm; 66-80min, 278nm; 80-90min, 322nm.

[0015] Preferably, the detection wavelength is 0-25 min, 280 nm; 25-66 min, 320 nm; 66-80 min, 280 nm; 80-90 min, 320 nm.

[0016] Preferably, the chromatographic column is selected from WATERS XSelect® HSS T3 C18 (4.6 mm × 250 mm, 5 μm) or Thermo Hypersil GOLD AQ (4.6 mm × 250 mm, 5 μm).

[0017] More preferably, the chromatographic column is selected from WATERS XSelect® HSS T3 C18 (4.6 mm × 250 mm, 5 μm).

[0018] Preferably, the mobile phase is: mobile phase A is acetonitrile, and mobile phase B is 0.1 wt% phosphoric acid solution.

[0019] Preferably, gradient elution is used, and the elution program is as follows:

[0020] Preferably, the flow rate in the high performance liquid chromatography is 0.95-1.1 mL / min.

[0021] The technical effects of this invention can be achieved by any point value or any sub-range value within the range of 0.95-1.1 mL / min, including but not limited to 0.95 mL / min, 1.0 mL / min, and 1.1 mL / min.

[0022] Preferably, the method for preparing the test solution includes the following steps: Take 1.0-2.0g of the sample to be tested, accurately weigh it, add 15-35mL of extraction solvent, weigh it, extract it, cool it, weigh it again, replenish the weight lost with the extraction solvent, shake it well, filter it, and take the filtrate to obtain the sample.

[0023] Preferably, the preparation method of the sample to be tested includes the following steps: weighing 11.19g of Rehmannia glutinosa, 5.60g of Adenophora stricta, 5.60g of Ophiopogon japonicus, 5.60g of Angelica sinensis, 11.19g of Lycium barbarum, and 7.46g of Melia toosendan (crushed), decocting twice, the first time for 60 minutes and the second time for 40 minutes, filtering, concentrating under reduced pressure, and freeze-drying to obtain the sample.

[0024] Preferably, the extraction solvent is selected from at least one of water, 20-80 wt% methanol solution, and 20-50 wt% ethanol solution.

[0025] More preferably, the extraction solvent is an 80 wt% methanol solution.

[0026] Preferably, the extraction is selected from at least one of ultrasound, heating reflux, or oscillation.

[0027] More preferably, the extraction is performed using ultrasound.

[0028] Preferably, the ultrasonic power is 380-420W, the frequency is 35-45kHz, and the duration is 20-40min.

[0029] The technical effects of this invention can be achieved by any point value or any sub-range within the range of 380-420W, including but not limited to: 380W, 381W, 382W, 383W, 384W, 385W, 386W, 387W, 388W, 389W, 390W, 391W, 392W, 393W, 394W, 395W, 396W, 397W, 398W, 399W, 400W, 401W, 402W, 403W, 404W, 405W, 406W, 407W, 408W, 409W, 410W, 411W, 412W, 413W, 414W, 415W, 416W, 417W, 418W, 419W, and 420W.

[0030] The technical effects of this invention can be achieved at any point value or any sub-range within the range of 35-45kHz, including but not limited to: 35kHz, 36kHz, 37kHz, 38kHz, 39kHz, 40kHz, 41kHz, 42kHz, 43kHz, 44kHz, and 45kHz.

[0031] The technical effects of this invention can be achieved at any point value or any sub-range within the range of 20-40 minutes, including but not limited to: 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, and 40 minutes.

[0032] More preferably, the ultrasonic power is 400W, the frequency is 40kHz, and the duration is 20min.

[0033] Preferably, the fingerprint pattern detection method includes the following steps: S1: Preparation of test solution: Take 1.0-2.0g of the sample to be tested, accurately weigh it, add 15-35mL of extraction solvent, weigh it, extract, cool it, weigh it again, replenish the weight lost with the extraction solvent, shake well, filter it, and take the filtrate to obtain the test solution.

[0034] S2: Preparation of reference solution: Weigh ferulic acid reference standard accurately, add extraction solvent to prepare a solution containing 10-14 μg per 1 mL; S3: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0035] More preferably, the fingerprint pattern detection method includes the following steps: S1: Take 1.5g of the sample to be tested, weigh it accurately, place it in a stoppered conical flask, add 25ml of 80wt% methanol accurately, weigh it, extract it by sonication at 400W power and 40kHz frequency for 20min, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain the sample.

[0036] S2: Preparation of reference solution: Accurately weigh ferulic acid reference standard, add extraction solvent to prepare a solution containing 12 μg per 1 mL; S3: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

[0037] Secondly, the present invention provides the application of the above-mentioned fingerprint spectrum detection method in the quality detection of traditional Chinese medicine.

[0038] The beneficial effects of this invention are as follows: 1. This invention studies the HPLC fingerprint of multiple active ingredients in Yiguanjian. Due to the complexity of the compound preparation, conventional high-performance liquid chromatography (HPLC) detection methods can often only detect one or a few components with high accuracy. The number of identifiable characteristic peaks is insufficient, and the coverage of characteristic components is low, making it difficult to achieve rapid, economical, and simultaneous quality control of multiple indicators on a conventional liquid chromatography platform. Therefore, this invention achieves efficient and accurate one-time detection of digitalisin C, 4-coumaric acid, ferulic acid, rutin, verbascoside, ligustilide I, and ligustilide in Yiguanjian through specific chromatographic conditions, including specific detection wavelengths, specific chromatographic columns, and specific gradient elution programs. This overcomes the shortcomings of existing technologies, and the method is simple and rapid to operate.

[0039] 2. The chromatographic column used in this invention is a conventional HPLC column, which is lower in cost and correspondingly cheaper in price than ultra-high pressure liquid chromatography columns. It is also more universal and easier to promote on a large scale.

[0040] 3. The specific detection method of this invention can effectively separate the peaks of various effective components in the decoction, with good separation degree and stable baseline. The methodological investigation results show that the detection method of this invention has high precision, stability, good repeatability, good robustness and low RSD value.

[0041] 4. This invention conducts a comparative study on the fingerprint spectrum of Yiguanjian (a traditional Chinese medicine formula), and identifies the specific absorption peaks of six medicinal materials—Rehmannia glutinosa, Adenophora stricta, Ophiopogon japonicus, Angelica sinensis, Lycium barbarum, and Melia toosendan—in the fingerprint spectrum. The established fingerprint spectrum also sets higher requirements for the quality control of the medicinal materials.

[0042] 5. This invention locates 12 common peaks in different batches of Yiguanjian decoction pieces and establishes a method for quantitative determination of these 12 common peaks using the standard curve method. After multiple verifications, it can be seen that the accuracy and repeatability meet the requirements. The determination method of this invention provides a reliable basis for the quality control of Yiguanjian and can be used for the quality control of the entire production process of Yiguanjian intermediates and granules in the future. Attached Figure Description

[0043] Figure 1 This is the HPLC chromatogram of the fingerprint detection method in Example 1.

[0044] Figure 2 This is the HPLC chromatogram of the fingerprint detection method in Example 2.

[0045] Figure 3 This is the HPLC chromatogram of the fingerprint detection method in Example 3.

[0046] Figure 4 This is the HPLC chromatogram of the fingerprint detection method in Example 4.

[0047] Figure 5 This is the HPLC chromatogram of the fingerprint detection method in Example 4.

[0048] Figure 6 For comparison with fingerprint patterns.

[0049] Figure 7 Fingerprint spectrum of Rehmannia glutinosa decoction alone.

[0050] Figure 8 Fingerprint spectrum of single decoction of North American ginseng.

[0051] Figure 9 Fingerprint spectrum of Ophiopogon japonicus decoction alone.

[0052] Figure 10 Fingerprint chromatogram of Angelica sinensis decoction alone.

[0053] Figure 11 Fingerprint spectrum of wolfberry decoction alone.

[0054] Figure 12 Fingerprint spectrum of single decoction of Sichuan Chinaberry.

[0055] Figure 13 This is the HPLC chromatogram of Comparative Example 1.

[0056] Figure 14 This is the HPLC spectrum of Comparative Example 2.

[0057] Figure 15 This is the HPLC spectrum of Comparative Example 3.

[0058] Figure 16 This is the HPLC chromatogram of Comparative Example 4.

[0059] Figure 17 This is the HPLC chromatogram for the precision test.

[0060] Figure 18 This is the HPLC chromatogram for the specificity test.

[0061] Figure 19 This is the HPLC chromatogram for the repeatability test.

[0062] Figure 20 This is the HPLC chromatogram for the intermediate precision test.

[0063] Figure 21 This is the HPLC chromatogram for the solution stability test.

[0064] Figure 22 This is an HPLC chromatogram for the durability test - extraction time.

[0065] Figure 23 HPLC chromatogram for durability test at column temperature.

[0066] Figure 24 HPLC chromatogram for durability test - flow rate.

[0067] Figure 25 HPLC spectrum for durability testing at the specified wavelength.

[0068] Figure 26 HPLC chromatogram of mobile phase phosphoric acid concentration for durability testing.

[0069] Figure 27 HPLC chromatogram of the column used in the durability test.

[0070] Note: Figure 17-27 In this context, "F" represents a peak.

[0071] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Detailed Implementation

[0072] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0073] Preparation of a consistent reference sample in this invention: Weigh out 11.19g of Rehmannia glutinosa, 5.60g of Adenophora stricta, 5.60g of Ophiopogon japonicus, 5.60g of Angelica sinensis, 11.19g of Lycium barbarum, and 7.46g of Melia toosendan (crushed). Decoction twice, the first time for 60 minutes and the second time for 40 minutes. Filter, concentrate under reduced pressure, and freeze dry to obtain the sample to be tested.

[0074] Example 1 A fingerprint detection method for a consistent standard sample S1: Preparation of test solution: Take 1.5g of the sample to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 80wt% methanol, weigh it, extract it by sonication at 400W power and 40kHz frequency for 20min, cool it, weigh it again, replenish the lost weight with 80wt% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0075] S2: Preparation of reference solution: Take an appropriate amount of ferulic acid reference standard, accurately weigh it, add extraction solvent to prepare a solution containing 12 μg per 1 mL; S3: Accurately pipette 10 μL each of the reference solution and the test solution into the high performance liquid chromatograph and determine them; The chromatographic conditions are as follows: Column: WATERS XSelect® HSS T3 C18 (4.6 mm × 250 mm, 5 μm); Column temperature: 30℃; Flow rate: 1 mL / min; Detection wavelengths: 0-25 min, 280 nm; 25-66 min, 320 nm; 66-80 min, 280 nm; 80-90 min, 320 nm; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is 0.1 wt% phosphoric acid solution; Gradient elution, the elution procedure is as follows:

[0076] The HPLC chromatogram of this embodiment is shown below. Figure 1 .from Figure 1 As can be seen from the results, the determination method of the present invention can effectively separate chromatographic peaks.

[0077] Example 2 Examination of extraction methods: Following the method described in Example 1, only the extraction method in step S1 was changed, and the impact of the extraction method on the detection results was examined: (1) Extract by shaking for 20 min; (2) Extract by reflux for 20 min; (3) Ultrasonic extraction: power 400W, frequency 40kHz, 20min.

[0078] The HPLC chromatogram of this embodiment is shown below. Figure 2 From top to bottom, the extraction methods are: shaking extraction, heating and reflux extraction, ultrasonic extraction, and blank sample (80wt% methanol). The chromatograms of the test samples obtained by the three extraction methods are basically the same. Therefore, ultrasonic extraction, which is simpler to operate, is preferred as the extraction method for determining the characteristic chromatogram of the standard sample.

[0079] Example 3 Investigation of extraction solvents: Following the method described in Example 1, only the extraction solvent in step S1 was changed, and the effect of the extraction solvent on the detection results was investigated: The extraction solvents were: water, methanol, ethanol, 20wt% methanol aqueous solution, 50wt% methanol aqueous solution, 80wt% methanol aqueous solution, 20wt% ethanol aqueous solution, 50wt% ethanol aqueous solution, and 80wt% ethanol aqueous solution.

[0080] The HPLC chromatogram of this embodiment is shown below. Figure 3 From top to bottom, the samples are: methanol, ethanol, 80wt% ethanol aqueous solution, 80wt% methanol aqueous solution, 50wt% ethanol aqueous solution, 20wt% ethanol aqueous solution, 20wt% methanol aqueous solution, water, and blank sample (80wt% methanol).

[0081] Experimental results show that the solvent effect is significant when methanol, ethanol, and 80wt% ethanol are used as solvents. The characteristic spectra of the test samples are basically consistent when other solvents are used. 80wt% methanol is preferred as the extraction solvent for the determination of the characteristic spectra of the standard decoction sample.

[0082] Example 4 Examination of extraction time: Following the method described in Example 1, only the extraction time in step S1 was changed, and the effect of the extraction solvent on the detection results was investigated: The extraction times were 20 min, 30 min, and 40 min, respectively.

[0083] The HPLC chromatogram of this embodiment is shown below. Figure 4 From top to bottom, the samples were 40 min, 30 min, 20 min, and a blank sample (80 wt% methanol).

[0084] Experimental results show that the characteristic spectra of the test samples obtained at different extraction times are basically consistent, and 20 minutes is the preferred extraction time for determining the characteristic spectra of the standard sample.

[0085] Example 5 Selection of detection wavelengths: Following the method described in Example 1, only the detection wavelength in step S3 was changed, and the effect of the detection wavelength on the detection results was examined: Within the range of 0-95 minutes, the detection wavelengths were 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, and 400nm, respectively.

[0086] The HPLC chromatogram of this embodiment is shown below. Figure 5 The wavelengths from top to bottom are 400nm, 390nm, 380nm, 370nm, 360nm, 350nm, 340nm, 330nm, 320nm, 310nm, 300nm, 290nm, 280nm, 270nm, 260nm, 250nm, 240nm, 230nm, 220nm, 210nm, and 200nm. It can be seen that in the 0-25 minute and 66-80 minute timeframes, the peak response values ​​in the chromatogram at 280nm are larger, and the baseline is relatively flat; in the 25-66 minute and 80-90 minute timeframes, the peak response values ​​in the chromatogram at 320nm are larger, and the baseline is relatively flat. Therefore, the wavelength switching is chosen between 280nm and 320nm.

[0087] Example 6 Fingerprint mapping 6.1 Fingerprint pattern Fifteen batches of reference samples were taken and test solutions were prepared according to Example 1. Using the chromatogram of sample 01 as a reference chromatogram, peak matching was performed using the median method and multi-point correction to finally generate the fingerprint chromatogram of the reference samples. Figure 6As shown. The results indicate that the chromatogram of the test solution should show 11 characteristic peaks. The peak corresponding to the reference peak (ferulic acid) in the reference solution is the S peak. The relative retention times of the remaining characteristic peaks and the S peak should be calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.29 (peak 1), 0.41 (peak 2), 0.60 (peak 3), 0.88 (peak 4), 0.92 (peak 5), 0.97 (peak 6), 1.04 (peak 8), 1.09 (peak 9), 1.20 (peak 10), and 1.48 (peak 11). Among them, peak 4: digitalisin C; peak 5: 4-coumaric acid; peak 7 (S): ferulic acid; peak 8: rutin; peak 9: verbascoside; peak 10: ligustilide I; and peak 11: ligustilide.

[0088] 6.2 Source of the main chromatographic peaks from medicinal materials Referring to the preparation of the test solution in step S1 of Example 1, test solutions of the six original medicinal materials of the prescription were prepared respectively. 10 μL of each solution was precisely pipetted and injected into the high-performance liquid chromatograph (HPLC). The chromatographic conditions described in Example 1 were used for determination. The results showed that the 11 common peaks in the standard fingerprint chromatogram of Yi Guan Jian were basically clearly identified in the fingerprint chromatogram of the prescription medicinal materials, including: Figure 7 As shown: Peaks 1, 4, 6, 7, and 9 originate from Rehmannia glutinosa; as Figure 8 As shown: Peak 7 originates from North American ginseng; as Figure 9 As shown: Peaks 5 and 12 originate from Ophiopogon japonicus; as Figure 10 As shown: Peaks 7, 10, and 11 are derived from Angelica sinensis; as Figure 11 As shown, peaks 1, 2, 3, 5, 7, and 8 are derived from wolfberries; as Figure 12 As shown: Peak 2 originates from Sichuan Chinaberry.

[0089] In summary, based on the study of the peak attribution in the characteristic spectral maps, we can conclude that: Peak 1 is mainly derived from wolfberry, with a small amount derived from rehmannia. Peak 2 is mainly derived from Sichuan pepper, with a small amount derived from wolfberry; Peak 3 is derived from wolfberry; Peak 4 is derived from Rehmannia glutinosa; Peak 5 is mainly derived from wolfberry, with a small amount from ophiopogon japonicus; Peak 6 originates from Rehmannia glutinosa; Peak 7 is mainly derived from Angelica sinensis, with a small amount derived from Glehnia littoralis, Lycium barbarum, and Rehmannia glutinosa; Peak 8 is derived from wolfberries; Peak 9 is derived from Rehmannia glutinosa; Peak 10 is derived from Angelica sinensis; Peak 11 is derived from Angelica sinensis; Peak 12 is derived from Ophiopogon japonicus (it was not included in the quality standard because the peak area of ​​Peak 12 is too small).

[0090] 6.3 A total of 15 batches of reference samples were prepared using qualified medicinal slices that met internal quality control requirements. These samples were then analyzed using the aforementioned characteristic spectral method. The relative retention times of the characteristic spectra are shown in Table 1. Table 1

[0091] As shown in Table 1, it meets the requirements of the "Technical Guidelines for the Study of Characteristic Spectra of Traditional Chinese Medicine Preparations (Trial)" and the relative retention times of each characteristic peak are all within the range of ±10% of the specified value.

[0092] Comparative Example 1 Compared with Example 1, only the gradient elution procedure in step S3 is changed as shown in Table 2 (gradient 1), and the rest is the same as in Example 1: Table 2

[0093] Comparative Example 2 Compared with Example 1, only the gradient elution procedure in step S3 is changed as shown in Table 3 (gradient 2), and the rest is the same as in Example 1: Table 3

[0094] Comparative Example 3 Compared with Example 1, only the gradient elution procedure in step S3 is changed as shown in Table 4 (gradient 3), and the rest is the same as in Example 1: Table 4

[0095] Comparative Example 4 Compared with Example 1, only the gradient elution procedure in step S3 is changed as shown in Table 5 (gradient 4), and the rest is the same as in Example 1: Table 5

[0096] The HPLC chromatograms of Comparative Examples 1-4 are as follows: Figures 13-16 As shown, gradients 1-3 exhibit incomplete peak information, with poor peak shape, separation, and overall distribution. Gradient 4, compared to other mobile phase gradients, provides more peak information, and demonstrates superior peak shape, separation, and overall distribution. However, no chromatographic peaks are observed between 70-85 minutes in the chromatogram, and the method analysis time is slightly long.

[0097] I. Methodological Examination: The detection method of Example 1 of this invention was used to conduct a methodological verification of the fingerprint spectrum of a traditional Chinese medicine. The selected evaluation indicators mainly included the following three aspects: precision, specificity, repeatability, intermediate precision, solution stability, and robustness test.

[0098] 1. Precision test Weigh appropriate amounts of ferulic acid reference standard and verbascoside reference standard, and gradually dilute with 80% methanol to prepare a solution containing approximately 12 μg of ferulic acid and approximately 5 μg of verbascoside per 1 mL. This is the reference solution. Inject the solution continuously according to the established procedure. The results are as follows: Figure 17 As shown.

[0099] The results showed that the theoretical plate number calculated based on the ferulic acid peak was no less than 5000; the retention time and peak area RSD of ferulic acid and verbascoside were no greater than 2.0% for six consecutive injections of reference solution, indicating that the system has good suitability and can be used for subsequent method validation.

[0100] 2. Specificity test Accurately pipette the reference solution (containing approximately 12 μg ferulic acid and 5 μg verbascoside per 1 mL), negative blank solution, and test solution, and continuously inject them according to the proposed analytical method. The results are as follows: Figure 18 As shown.

[0101] The results showed that the negative blank solution had no interference; the retention times of the two chromatographic peaks in the test sample chromatogram should correspond to those in the reference sample chromatogram; and the method had good specificity.

[0102] 3. Repeatability test Take an appropriate amount of dried powder from a traditional Chinese medicine decoction, grind it into a fine powder, and prepare six parallel test solutions. Analyze the solutions continuously by injection according to the proposed analytical method. The results are as follows: Figure 19 As shown.

[0103] The results showed that the RSD of the relative retention time of each characteristic peak in the six test samples was no greater than 5.0%, indicating that the method had good repeatability.

[0104] 4. Intermediate precision test The intermediate precision of this method was assessed by parallel determination of the relative retention times of six (12) samples of Yiguanjian granules at different times using different high-performance liquid chromatographs by different personnel. The samples were analyzed continuously using the proposed analytical method, and the results are as follows: Figure 20 As shown.

[0105] The results showed that the RSD values ​​of the relative retention times of each characteristic peak in the 12 test samples were all no greater than 5.0%, indicating that the intermediate precision of the method was good.

[0106] 5. Solution stability test At 0, 10, 24, 48, and 72 hours, the test solution was precisely pipetted and injected according to the proposed analytical method. The analysis was performed continuously using the proposed analytical method, and the results are as follows: Figure 21 As shown.

[0107] Compared with 0h, the relative retention time (RAD) of each characteristic peak in the test sample was no greater than 5.0%, indicating that the test sample solution was stable within 72h.

[0108] 6. Durability test (1) Extraction time Following the method described in Example 1, only the extraction time in step S1 was changed to 18 min, 20 min, and 22 min, and the results are as follows: Figure 22 As shown.

[0109] The results showed that, compared with the original conditions, the relative retention time (RAD) of each characteristic peak in the sample was no greater than 5.0%, indicating that the method had good robustness under small changes in extraction time.

[0110] (2) Column temperature Following the method described in Example 1, only the column temperature in step S3 was changed to 28°C, 30°C, and 32°C, and the results were as follows: Figure 23 As shown.

[0111] The results showed that, compared with the original conditions, the relative retention time (RAD) of each characteristic peak in the test sample was no greater than 5.0%, indicating that the method had good robustness under small changes in column temperature.

[0112] (3) Flow velocity Following the method described in Example 1, only the flow rate in step S3 was changed to 0.9 mL / min, 0.95 mL / min, 1 mL / min, and 1.1 mL / min, and the results were as follows. Figure 24 As shown.

[0113] The results showed that for a flow rate variation of 1.0 ml / min ± 0.1 ml / min, when the flow rate was 0.9 ml / min, the peak elution time of peak 2 was exactly at the wavelength switching point of 280 / 320 nm, resulting in incomplete detection of the chromatographic peak. Therefore, the flow rate range was narrowed to 0.95 / 1.0 / 1.1 ml / min. The relative retention time (RAD) values ​​of each characteristic peak in the chromatogram of the test sample were all no greater than 5.0%, indicating good robustness.

[0114] (4) Wavelength Following the method described in Example 1, only the wavelength in step S3 was changed to ±2 nm of the original wavelength, and the result is as follows: Figure 25 As shown.

[0115] The results showed that, compared with the original conditions, under slight changes in the detection wavelength, the relative retention time (RAD) of each characteristic peak in the chromatogram of the test sample was no greater than 5.0%, indicating good robustness.

[0116] (5) Concentration of phosphoric acid in the mobile phase Following the method described in Example 1, only the phosphoric acid concentration of mobile phase B in step S3 was changed to 0.09 wt%, 0.1 wt%, and 0.11 wt%, and the results were as follows. Figure 26 As shown.

[0117] The results showed that, compared with the original conditions, under slight changes in the concentration of phosphoric acid in the mobile phase, the relative retention time (RAD) of each characteristic peak in the chromatogram of the test sample was no greater than 5.0%, indicating good robustness.

[0118] (6) Chromatographic column Following the method described in Example 1, only the batch number of the chromatographic column or the brand of the packing material in step S3 was changed, and the result was as follows: Figure 27 As shown.

[0119] Example 1: The original chromatographic column was a WATERS XSelect® HSS T3 C18 column (4.6 mm × 250 mm, 5 μm, Part. No. 186004793, Ser. No. 02113511413601, LOT 0211351141).

[0120] For different batches of the same brand, WATERS XSelect® HSS T3 C18 columns (4.6 mm × 250 mm, 5 μm, Part. No. 186004793, Ser. No. 02133514214004, LOT 0213351421) (column A), the RAD values ​​of the relative retention times of each characteristic peak were no greater than 5.0%. However, for Thermo Hypersil GOLDAQ columns (4.6 mm × 250 mm, 5 μm, Part. No. 25005-254630, Ser. No. 20274857, LOT 20215) (column B) with the same packing material, the RAD values ​​of the relative retention times of each characteristic peak ranged from 0.3% to 14.3%. Therefore, the column should be fixed when using this method.

[0121] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting fingerprint spectra of a standard sample, characterized in that, The test solution was analyzed by high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: Chromatographic column: C18 column; Column temperature: 28℃-32℃; Detection wavelengths: 0-25 min, 278-282 nm; 25-66 min, 318-322 nm; 66-80 min, 278-282 nm; 80-90 min, 318-322 nm; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a 0.09-0.11 wt% phosphoric acid solution; Gradient elution, the elution procedure is as follows: 。 2. The fingerprint pattern detection method according to claim 1, characterized in that, The detection wavelengths are 0-25 min, 280 nm; 25-66 min, 320 nm; 66-80 min, 280 nm; and 80-90 min, 320 nm.

3. The fingerprint pattern detection method according to claim 1, characterized in that, The chromatographic column is selected from WATERSXSelect® HSS T3 C18 or Thermo Hypersil GOLD AQ.

4. The fingerprint pattern detection method according to claim 3, characterized in that, The chromatographic column was selected from WATERSXSelect® HSS T3 C18.

5. The fingerprint pattern detection method according to claim 1, characterized in that, Gradient elution, the elution procedure is as follows: 。 6. The fingerprint pattern detection method according to claim 1, characterized in that, The flow rate for the high performance liquid chromatography is 0.95-1.1 mL / min.

7. The fingerprint pattern detection method according to claim 1, characterized in that, The method for preparing the test solution includes the following steps: Take 1.0-2.0g of the sample to be tested, accurately weigh it, add 15-35mL of extraction solvent, weigh it, extract it, cool it, weigh it again, replenish the weight lost with the extraction solvent, shake it well, filter it, and take the filtrate to obtain the sample.

8. The fingerprint pattern detection method according to claim 7, characterized in that, The extraction solvent is selected from at least one of water, 20-80wt% methanol solution, and 20-50wt% ethanol solution; the extraction is selected from at least one of ultrasound, heating reflux, or oscillation; the ultrasound power is 380-420W, the frequency is 35-45kHz, and the time is 20-40min.

9. The fingerprint pattern detection method according to claim 1, characterized in that, The fingerprint pattern detection method includes the following steps: S1: Preparation of test solution: Take 1.0-2.0g of the sample to be tested, accurately weigh it, add 15-35mL of extraction solvent, weigh it, extract, cool it, weigh it again, replenish the weight lost with the extraction solvent, shake well, filter it, and take the filtrate to obtain the test solution. S2: Preparation of reference solution: Weigh ferulic acid reference standard accurately, add extraction solvent to prepare a solution containing 10-14 μg per 1 mL; S3: Accurately pipette 10 μL each of the reference solution and the test solution into the high-performance liquid chromatograph and determine the result.

10. The application of the fingerprint spectrum detection method according to any one of claims 1-9 in the quality detection of a traditional decoction.

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