A method for constructing a gastrointestinal an pill characteristic spectrum and application thereof

Abstract

The application specifically relates to a construction method of a Weichang'an pill characteristic spectrum and application thereof, and belongs to the technical field of traditional Chinese medicine analysis. The method comprises preparation of a test sample solution and a reference solution, high performance liquid chromatography is adopted, acetonitrile-0.1% phosphoric acid is used as a mobile phase to perform specific gradient elution, and a double-wavelength switching technology (0-15 min is 290 nm, and 15-83 min is 225 nm) is combined to construct a characteristic spectrum containing 19 common peaks. Tiglioside, naringin and honokiol are used as reference peaks to determine the relative retention time range of each peak. Through methodological verification, the method is simple in operation, good in repeatability, high in precision and strong in stability, can comprehensively and accurately reflect the overall appearance of non-volatile components in the Weichang'an pill, can objectively evaluate the quality uniformity of the product, and provides a new method for quality control of the preparation.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for constructing a characteristic spectrum of Weichangan Pill and its application. Background Technology

[0002] Weichangan Pills are a traditional Chinese medicine produced by Lerentang Pharmaceutical Factory of Tianjin Darentang Group Co., Ltd. It consists of ten medicinal ingredients: costus root, sandalwood, agarwood, rhubarb, magnolia bark (processed with ginger), immature bitter orange (stir-fried with wheat bran), chuanxiong rhizome, croton oil, jujube, and artificial musk. It has the effects of aromatic purification, regulating qi and relieving pain, and strengthening the stomach and promoting digestion. Clinically, it is used to treat diarrhea, enteritis, bacillary dysentery, abdominal distension, food stagnation and milk stagnation caused by indigestion.

[0003] Currently, the quality standard for Weichang'an Pills is included in Part I of the 2025 edition of the Chinese Pharmacopoeia, mainly including its properties, thin-layer chromatography identification, routine examination, and the determination of the content of magnolol and honokiol. In existing technologies, most disclosed quality control methods focus on the determination of the content of a single indicator component. For example, authorized patent CN102507756B discloses a method for determining the content of ferulic acid in Ligusticum chuanxiong and emodin and chrysophanol in rhubarb, but this method is still a "point-to-point" control and cannot comprehensively reflect the overall chemical composition information of the compound preparation. Regarding volatile components, authorized patent CN113984917B discloses a method for detecting volatile components in Weichang'an Pills using gas chromatography-mass spectrometry (GC-MS), and constructs a volatile characteristic spectrum, providing an effective means for the quality control of its volatile components.

[0004] However, the prescription for Weichangan Pills is complex, and its efficacy is the result of the synergistic effect of multiple components. The existing technologies mentioned above focus on the content of non-volatile indicator components and the characteristic spectra of volatile components, respectively, and have not yet established a method for controlling the overall characteristic spectra of non-volatile, highly polar components. Single content determination cannot characterize changes in unknown components, and GC-MS characteristic spectra only target volatile components, failing to effectively monitor the numerous non-volatile, thermally unstable, or highly polar components present in the formula (such as crotonin, flavonoid glycosides, and anthraquinone glycosides).

[0005] Therefore, establishing a characteristic map construction method that can comprehensively and stably characterize the overall appearance of the non-volatile components of Weichangan Pills is of great significance for improving its quality control system and ensuring product quality uniformity and clinical efficacy stability. Summary of the Invention

[0006] To address the technical challenge of existing technologies that rely solely on content determination and volatile characterization to effectively monitor non-volatile, thermally unstable, or highly polar components in Weichang'an pills, this invention provides a method for constructing characteristic chromatograms that comprehensively and stably characterize the overall appearance of its non-volatile components, along with its application. This method, through optimization of chromatographic conditions and detection wavelengths, achieves the simultaneous separation and characterization of multiple non-volatile components in Weichang'an pills, providing a new means to improve its quality control system.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention discloses a method for constructing a characteristic map of a gastrointestinal soothing pill, comprising the following steps:

[0009] (1) Preparation of the test solution;

[0010] (2) Preparation of the reference solution;

[0011] (3) The reference solution and the test solution were determined by high performance liquid chromatography, and characteristic chromatograms were constructed.

[0012] The chromatographic conditions are as follows:

[0013] Octadecylsilane-bonded silica gel is used as a filler;

[0014] Using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the following procedure:

[0015] From 0 to 10 minutes, mobile phase A changes from 3% to 10%, and mobile phase B changes from 97% to 90%.

[0016] Within 10–42 minutes, mobile phase A changed from 10% to 26%, and mobile phase B changed from 90% to 74%.

[0017] From 42 to 83 minutes, mobile phase A decreased from 26% to 76%, and mobile phase B decreased from 74% to 24%.

[0018] Detection was performed using wavelength switching technology; the detection wavelength was 290 nm for 0–15 minutes and 225 nm for 15–83 minutes.

[0019] The column temperature was 35℃; the flow rate was 1.0 mL / min.

[0020] As a preferred embodiment of the present invention, the reference solution in step (2) is a mixed solution containing crotonin, naringin, and magnolol. More preferably, each 1 mL of the reference solution contains 0.03 mg of crotonin, 0.3 mg of naringin, and 0.3 mg of magnolol.

[0021] As a preferred embodiment of the present invention, the preparation method of the test solution in step (1) is as follows: take the sample of Weichangan pills, grind it into a fine powder, accurately add 20 mL of 70% methanol solution, weigh it, sonicate it for 30 minutes, cool it, weigh it again, replenish the lost weight with 70% methanol solution, shake it well, filter it, and take the filtrate to obtain the test solution.

[0022] As a preferred embodiment of the present invention, the ultrasonic extraction power in step (1) is 250W and the frequency is 40kHz.

[0023] As a preferred embodiment of the present invention, the high performance liquid chromatography method uses an OSAKA SODACAPCELL PAK C18 MGⅡ column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0024] Secondly, the present invention provides a characteristic spectrum of the gastrointestinal analgesic pill constructed by the above method. The characteristic spectrum has 19 common characteristic peaks, wherein peak 1 is the S1 peak corresponding to the crotonin reference peak, peak 9 is the S2 peak corresponding to the naringin reference peak, and peak 14 is the S3 peak corresponding to the honokiol reference peak.

[0025] As a preferred embodiment of the present invention, the relative retention time of each characteristic peak and the S peak is within ±10% of a specified value;

[0026] The specified relative retention times with peak S1 are: 1.26 for peak 2 and 1.63 for peak 3;

[0027] The specified relative retention times with peak S2 are as follows: peak 4 is 0.54, peak 5 is 0.78, peak 6 is 0.84, peak 7 is 0.96, peak 8 is 0.97, peak 10 is 1.03, and peak 11 is 1.07.

[0028] The specified relative retention times with peak S3 are as follows: 0.93 for peak 12, 0.99 for peak 13, 1.01 for peak 15, 1.03 for peak 16, 1.04 for peak 17, 1.05 for peak 18, and 1.09 for peak 19.

[0029] As a preferred embodiment of the present invention, the medicinal materials with the common characteristic peaks are classified as follows:

[0030] Peak 1 comes from croton oil; Peaks 2-3 come from jujube; Peaks 4 and 12 come from Sichuan lovage rhizome; Peak 5 comes from agarwood; Peak 6 comes from Sichuan lovage rhizome; Peak 7 comes from bitter orange peel; Peak 8 comes from sandalwood; Peaks 9-11 come from bitter orange peel; Peaks 13 and 19 come from rhubarb; Peaks 14, 17, and 18 come from magnolia bark; Peaks 15-16 come from costus root.

[0031] Thirdly, the present invention provides an application of the above-mentioned characteristic spectrum of gastrointestinal analgesia pills in the quality detection of gastrointestinal analgesia pills, comprising the following steps:

[0032] (1) The chromatogram of the sample to be tested should show 19 characteristic peaks;

[0033] (2) The relative retention time of each characteristic peak and the S peak should be within ±10% of the specified value;

[0034] (3) Import the chromatogram of the sample to be tested into the similarity evaluation system of chromatographic feature chromatogram of traditional Chinese medicine, and calculate the similarity with the aforementioned feature chromatogram of Weichangan Pill. The similarity should not be less than 0.90.

[0035] The specified value is:

[0036] Relative retention times with peak S1: peak 2 is 1.26, peak 3 is 1.63;

[0037] Relative retention times with S2 peak: peak 4 is 0.54, peak 5 is 0.78, peak 6 is 0.84, peak 7 is 0.96, peak 8 is 0.97, peak 10 is 1.03, and peak 11 is 1.07;

[0038] Relative retention times with peak S3: 0.93 for peak 12, 0.99 for peak 13, 1.01 for peak 15, 1.03 for peak 16, 1.04 for peak 17, 1.05 for peak 18, and 1.09 for peak 19.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) This invention establishes for the first time a method for constructing the overall characteristic spectrum of non-volatile highly polar components of Weichangan pills, which complements the existing single component content determination and volatile component characteristic spectrum (GC-MS) well, makes up for the shortcomings of the prior art, and provides a more comprehensive quality control means for this preparation.

[0041] (2) This invention achieves systematic optimization of chromatographic conditions and maximizes the acquisition of component information. Through extensive screening, the optimal gradient elution program was determined, and a dual-wavelength switching detection technology (0–15 min / 290 nm, 15–83 min / 225 nm) was innovatively adopted, enabling good response and separation of chromatographic peaks of chemical components with different properties, thus maximizing characteristic peak information. At the same time, crotonin, naringin, and magnolol were used as references to represent chemical components with different polarity ranges (S1, S2, S3), effectively controlling the drift of chromatographic peaks throughout the time period and significantly improving the stability and comparability of characteristic spectra.

[0042] (3) The method of this invention is simple to operate. Methodological verification shows that the RSD of the relative retention times of each characteristic peak in repeatability, precision, and stability tests is less than 2%, indicating that the method has good repeatability, high precision, and strong stability, and can meet the quality control requirements of complex traditional Chinese medicine systems. The constructed characteristic chromatograms can comprehensively and accurately reflect the overall chemical composition of the product, objectively evaluate its process rationality and quality uniformity, and provide a reliable new method to ensure the safety and efficacy of clinical medication. Attached Figure Description

[0043] Figure 1 The chromatograms of the gastrointestinal analgesia pill test solution under different gradient elution systems are compared. From top to bottom, the chromatograms are as follows: System 1 (225 nm), System 1 (290 nm), System 2 (225 nm), System 2 (290 nm), System 3 (225 nm), System 3 (290 nm), System 4 (225 nm), System 4 (290 nm), System 5 (225 nm), System 5 (290 nm), System 6 (225 nm), System 6 (290 nm).

[0044] Figure 2 Three-dimensional full-scan spectrum (200-400 nm) of the diode array detector for the test solution of Weichangan Pills. The horizontal axis represents retention time (min), the vertical axis represents wavelength (nm), and the color intensity represents absorption intensity.

[0045] Figure 3 The chromatograms of the gastrointestinal analgesia pill test solution at different detection wavelengths are compared. From top to bottom, the wavelengths are: 225nm, 254nm, 283nm, and 290nm.

[0046] Figure 4 A comparison of chromatograms of the gastrointestinal analgesia pill test solution under different chromatographic columns is shown. From top to bottom, the columns are: Zafex Supperfex JX-C18 column, SHIMADZU Shim-pack GIST C18-AQ column, SHIMADZU GLSciences Inertsil ODS-3 column, OSAKA SODA CAPCELL PAK MG column, OSAKA SODA CAPCELL PAK UG120 column, and OSAKA SODA CAPCELL PAK C18 MGⅡ column.

[0047] Figure 5 The chromatograms of the gastrointestinal analgesia pill test solution at different column temperatures are compared. From top to bottom, the chromatograms are: 30℃, 35℃, and 40℃.

[0048] Figure 6The chromatograms of the gastrointestinal analgesia pill test solution at different flow rates are compared. From top to bottom, the flow rates are: 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min.

[0049] Figure 7 Comparison of delayed chromatograms of the test solution of Weichangan Pills. From top to bottom in the figure: the test solution with doubled retention time, and the test solution with normal retention time.

[0050] Figure 8 The chromatograms of the gastrointestinal analgesia pill test solution under different extraction methods are compared. From top to bottom, the chromatograms are: reflux, ultrasonication;

[0051] Figure 9 The chromatograms of the gastrointestinal analgesia pill test solution under different extraction solvents are compared. From top to bottom, the chromatograms are: methanol, 70% methanol, 50% methanol, 30% methanol, and water.

[0052] Figure 10 The chromatograms of the gastrointestinal analgesia pill test solution at different extraction times are compared. From top to bottom, the chromatograms represent: 30 minutes, 60 minutes, and 90 minutes.

[0053] Figure 11 The chromatograms of the gastrointestinal analgesia pill test solution under different extraction solvent amounts are compared. From top to bottom in the figure, they are: 15 mL, 20 mL, and 50 mL.

[0054] Figure 12 This is a chromatogram of the reference solution. From left to right in the figure are: crotonin, naringin, and magnolol.

[0055] Figure 13 The chromatogram of the test solution for Weichangan Pills;

[0056] Figure 14 The chromatogram is for the blank solvent;

[0057] Figure 15 The chromatograms are for all batches of the test solution of Weichangan Pills. The batch numbers in the figure from top to bottom are: 1531492, 1531502, 1531515, 1531522, 1531533, 1531543, 1531556, 1531567, 1531575, 1531584, 1531592, 1531606, 1531614, 1531619, 1531622, and 1531613.

[0058] Figure 16 The chromatogram of the gastrointestinal analgesia pill with peak positions marked;

[0059] Figure 17The chromatograms for the classification analysis of croton oil are shown below. From top to bottom, they are: chromatogram of croton oil reference material, chromatogram of crotonin reference standard, chromatogram of gastrointestinal analgesia pill test sample, and chromatogram of croton oil-deficient negative sample.

[0060] Figure 18 The chromatograms for the classification analysis of jujube medicinal materials are shown from top to bottom as follows: chromatogram of jujube reference medicinal material, chromatogram of gastrointestinal analgesia pill test sample, and chromatogram of negative sample lacking jujube.

[0061] Figure 19 The chromatograms for the classification analysis of Ligusticum chuanxiong are shown below. From top to bottom, they are the chromatograms of Ligusticum chuanxiong reference material, ferulic acid reference standard, gastrointestinal analgesia pill test sample, and negative sample lacking Ligusticum chuanxiong.

[0062] Figure 20 The chromatograms for the attribution analysis of Magnolia officinalis are as follows: from top to bottom, the chromatograms of Magnolia officinalis reference material, magnolol reference standard, magnolol reference standard, gastrointestinal analgesia pill test sample, and negative sample lacking Magnolia officinalis.

[0063] Figure 21 The chromatograms for the classification analysis of rhubarb medicinal material are shown below. From top to bottom, they are: chromatogram of rhubarb reference material, chromatogram of emodin reference standard, chromatogram of chrysophanol reference standard, chromatogram of gastrointestinal analgesia pill test sample, and chromatogram of negative sample lacking rhubarb.

[0064] Figure 22 The chromatograms for the attribution analysis of artificial musk are shown below. From top to bottom, they are the chromatograms of the artificial musk reference material, the gastrointestinal analgesia pill test sample, and the negative sample lacking artificial musk.

[0065] Figure 23 The chromatograms for the classification analysis of agarwood are shown from top to bottom as follows: agarwood reference material, agarwood tetraol reference standard, gastrointestinal analgesic pill test sample, and a negative sample lacking agarwood.

[0066] Figure 24 The chromatograms for the classification analysis of Citrus aurantium are as follows: from top to bottom, the chromatograms are of Citrus aurantium reference material, rutin reference standard, naringin reference standard, neohesperidin reference standard, hesperidin reference standard, gastrointestinal analgesic pill test sample, and negative sample lacking Citrus aurantium.

[0067] Figure 25 The chromatograms for the attribution analysis of sandalwood are shown below, from top to bottom: chromatogram of sandalwood reference material, chromatogram of gastrointestinal analgesia pill test sample, and chromatogram of negative sample lacking sandalwood.

[0068] Figure 26The chromatograms for the classification analysis of Costus root are as follows: from top to bottom, the chromatograms are: Costus root reference material, Costus root hydrocarbon lactone reference standard, dehydrocostus root lactone reference standard, Gastrointestinal An Pill test sample, and Costus root-deficient negative sample.

[0069] Figure 27 The chromatograms of the relevant reference standards are shown below, from top to bottom: crotonin reference standard, linalool reference standard, ferulic acid reference standard, rutin reference standard, naringin reference standard, hesperidin reference standard, neohesperidin reference standard, emodin reference standard, magnolol reference standard, costunolide reference standard, dehydrocostunolide reference standard, magnolol reference standard, and rhein reference standard.

[0070] Figure 28 The chromatograms of the relevant reference medicinal materials are shown below, from top to bottom: Croton tiglium powder, Jujube, Ligusticum chuanxiong, Agarwood, Citrus aurantium, Sandalwood, Rhubarb, Magnolia officinalis, Aucklandia lappa, and Artificial musk.

[0071] Figure 29 The image shows the chromatogram of Weichangan Pills, batch number 1531613. From top to bottom, these are the chromatograms of 6 samples from the same batch.

[0072] Figure 30 This is a chromatogram overlay for precision testing. From top to bottom, the chromatograms are the chromatograms of the test solution obtained from 6 consecutive injections (precision 1 to 6).

[0073] Figure 31 This is a chromatogram overlay for stability testing. From top to bottom, the chromatograms are obtained from the determination of the test solution after being placed at room temperature for 0, 6, 12, 18, and 24 hours.

[0074] Figure 32 The chromatograms of the gastrointestinal analgesia pill test solution under different chromatographic column conditions are shown below. The batches from top to bottom are A4AD 24377, A4AD 02474, and A4AD 50811.

[0075] In the attached diagram, peak 1 (S1): crotonin, peak 2: jujube component 1, peak 3: jujube component 2, peak 4: chuanxiong component 1, peak 5: agaricol, peak 6: ferulic acid, peak 7: rutin, peak 8: sandalwood component, peak 9 (S2): naringin, peak 10: hesperidin, peak 11: neohesperidin, peak 12: chuanxiong component 2, peak 13: emodin, peak 14 (S3): magnolol, peak 15: costus lactone, peak 16: dehydrocostus lactone, peak 17: magnolia officinalis component, peak 18: magnolol, peak 19: emodin. Detailed Implementation

[0076] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0077] After reading the contents disclosed in this invention, those skilled in the art can make appropriate adjustments or substitutions to the process parameters of the methods and applications described in this invention without departing from the spirit and scope of this invention. Such obvious adjustments, substitutions or combinations should be included within the protection scope of this invention.

[0078] Unless otherwise specified, the materials, reagents, instruments and testing methods used in the following embodiments can be obtained commercially or prepared, operated and implemented with reference to conventional methods disclosed in the art.

[0079] It should be noted that all technical parameters described in this document as numerical ranges (such as temperature, ratio, time, content, etc.) should be understood as encompassing all possible sub-ranges and specific numerical points within that range, regardless of whether the specific numerical value or sub-range is explicitly listed. Unless otherwise specified, the technical terms used in this document have the meanings commonly understood by those skilled in the art.

[0080] Example 1: Construction of the characteristic map of Gastrointestinal Comfort Pills

[0081] 1. Instruments and reagents

[0082] Instruments: SHIMADZU LC-20AD high performance liquid chromatograph (equipped with diode array detector and LabSolutions workstation); Agilent 1260 high performance liquid chromatograph (equipped with ChemStation workstation); METTLER TOLEDOXS205DU electronic analytical balance; AS10200AT ultrasonic cleaner (power 250W, frequency 40kHz); DZKW-4 electric thermostatic water bath.

[0083] Chromatographic column: OSAKA SODA CAPCELL PAK C18 MGⅡ (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm, batch number: A4AD 50811); the effects of different batches of the same brand (batch numbers: A4AD 24377, A4AD 02474) and different brands of columns (Zafex Supperfex JX-C18, batch number WX0101EL94; SHIMADZU Shim-packGIST C18-AQ, batch number 20L19205; SHIMADZU GL Sciences Inertsil ODS-3, batch number 1A7145260; OSAKA SODA CAPCELL PAK MG, batch number AKAD50464; OSAKA SODA CAPCELL PAK UG120, batch number AOAD48389) on the separation performance were also investigated.

[0084] Reference standards: Croton tigrin (batch number 111856-201102), Naringin (batch number 110722-202417), Magnolol (batch number 110730-202416), Ferulic acid (batch number 110773-202316), Rhein (batch number 110796-202423), Emodin (batch number 110756-202414), Hesperidin (batch number 1107) The following reference standards were purchased from the National Institutes for Food and Drug Control: 21-202220), neohesperidin (batch number 111857-202305), magnolol (batch number 110729-202316), costunolide (batch number 111524-202312), and dehydrocostunolide (batch number 111525-202313); rutin (batch number FS1622044) was purchased from Tianjin Alta Technology Co., Ltd.

[0085] Reference medicinal materials: Croton oil, jujube, chuanxiong rhizome, agarwood, sandalwood, immature bitter orange peel, rhubarb, magnolia bark, and costus root were all purchased from the China National Institutes for Food and Drug Control, with batch numbers of jujube 121040-202410, chuanxiong rhizome 120918-201813, agarwood 121222-202104, sandalwood 121240-201703, immature bitter orange peel 120981-202106, rhubarb 120984-202203, magnolia bark 121285-202304, and costus root 120921-202511; artificial musk reference medicinal material was provided by Lerentang Pharmaceutical Factory of Tianjin Darentang Group Co., Ltd.

[0086] Reagents: Acetonitrile was chromatographic grade (Merck), phosphoric acid was analytical grade (Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.), methanol was chromatographic grade (Merck), and water was Milli-Q ultrapure water.

[0087] Samples: A total of 16 batches of Weichangan Pills were collected, with batch numbers 1531492, 1531502, 1531515, 1531522, 1531533, 1531543, 1531556, 1531567, 1531575, 1531584, 1531592, 1531606, 1531614, 1531619, 1531622, and 1531613, provided by Lerentang Pharmaceutical Factory of Tianjin Darentang Group Co., Ltd.

[0088] 2. Screening of chromatographic conditions

[0089] 2.1 Selection of mobile phase elution program

[0090] To achieve optimal separation, the gradient elution program was systematically optimized in this invention. Acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, the flow rate was 1.0 mL / min, and the detection wavelengths were set to 225 nm and 290 nm, respectively. Six different gradient elution programs (System 1 to System 6) were compared to determine the optimal chromatographic separation conditions.

[0091] System 1: 0–10 min, mobile phase A 5%→10%, mobile phase B 95%→90%; 10–11 min, mobile phase A 10%→20%, mobile phase B 90%→80%; 11–45 min, mobile phase A 20%, mobile phase B 80%; 45–70 min, mobile phase A 20%→90%, mobile phase B 80%→10%; 70–75 min, mobile phase A 90%, mobile phase B 10%.

[0092] System 2: 0–5 min, mobile phase A 10%, mobile phase B 90%; 5–6 min, mobile phase A 10%→16%, mobile phase B 90%→84%; 6–30 min, mobile phase A 16%→25%, mobile phase B 84%→75%; 30–60 min, mobile phase A 25%→85%, mobile phase B 75%→15%; 60–70 min, mobile phase A 85%, mobile phase B 15%.

[0093] System 3: 0–45 min, mobile phase A 3%→40%, mobile phase B 97%→60%; 45–80 min, mobile phase A 40%→80%, mobile phase B 60%→20%.

[0094] System 4: 0–10 min, mobile phase A 3%→10%, mobile phase B 97%→90%; 10–10.10 min, mobile phase A 10%→15%, mobile phase B 90%→85%; 10.10–40 min, mobile phase A 15%→35%, mobile phase B 85%→65%; 40–80 min, mobile phase A 35%→90%, mobile phase B 65%→10%.

[0095] System 5: 0–10 min, mobile phase A 3%→10%, mobile phase B 97%→90%; 10–35 min, mobile phase A 10%→34%, mobile phase B 90%→67%; 35–75 min, mobile phase A 34%→80%, mobile phase B 67%→20%.

[0096] System 6: 0–10 min, mobile phase A 3%→10%, mobile phase B 97%→90%; 10–42 min, mobile phase A 10%→26%, mobile phase B 90%→74%; 42–83 min, mobile phase A 26%→76%, mobile phase B 74%→24%.

[0097] Depend on Figure 1 It is evident that Systems 1 through 5 suffer from varying degrees of problems, including uneven peak distribution, poor resolution, and overlap between target and impurity peaks. In contrast, System 6 exhibits uniform peak distribution across all detection wavelengths, good resolution between the main component peak and adjacent peaks, moderate relative retention times for each characteristic peak, symmetrical peak shapes, and rich overall chromatographic information. Therefore, System 6 is determined to be the optimal gradient elution program of this invention.

[0098] 2.2 Selection of detection wavelength

[0099] A three-dimensional full-wavelength scan of the gastrointestinal analgesia pill test solution was performed using a diode array detector. The results are shown in the figure. Figure 2 .Depend on Figure 2 It was found that the main chromatographic peaks exhibited good UV absorption responses at wavelengths of 225 nm and 290 nm. Specifically, the peak response values ​​at 225 nm were higher, which is beneficial for the detection of low-content components. At 290 nm, some characteristic peaks (such as naringin and magnolol) showed characteristic absorption, which could be effectively distinguished from other impurity peaks. Further comparison of chromatograms at different wavelengths is shown in the figure. Figure 3 In the 0–15 minute time period, the number and intensity of chromatographic peaks at 290 nm wavelength were superior to those at other wavelengths; in the 15–83 minute time period, the number and intensity of peaks at 225 nm wavelength were significantly superior to those at other wavelengths.

[0100] Taking into account the response intensity and characteristics of each chromatographic peak, this invention employs wavelength switching technology for detection, specifically using a detection wavelength of 290 nm for 0–15 minutes and 225 nm for 15–83 minutes. This setting can accommodate the ultraviolet absorption characteristics of chemical components with different polarities, obtaining the richest chromatographic information and enabling simultaneous monitoring and characteristic chromatogram construction of multiple components of the gastrointestinal analgesic pill.

[0101] 2.3 Selection of Chromatographic Column

[0102] The effects of different brands and different specifications of the same brand of chromatographic columns on separation performance were investigated. Six chromatographic columns were selected: Zafex Supperfex JX-C18, SHIMADZU Shim-pack GIST C18-AQ, SHIMADZU GL Sciences Inertsil ODS-3, OSAKA SODA CAPCELL PAK MG, OSAKA SODA CAPCELL PAK UG120, and OSAKA SODA CAPCELL PAK C18 MGⅡ (all with specifications of 4.6 mm × 250 mm and 5 μm), and experiments were conducted under the proposed chromatographic conditions.

[0103] Figure 4 The results showed that different chromatographic columns exhibited varying retention behaviors and resolutions for the components in the sample. Specifically, the OSAKA SODA CAPCELL PAK MGⅡ column demonstrated moderate retention times for each characteristic peak, symmetrical peak shapes, and good resolution with adjacent peaks, meeting the requirements for characteristic chromatogram analysis. Therefore, the OSAKA SODA CAPCELL PAK C18 MGⅡ column was selected as the column used in this method.

[0104] 2.4 Investigation of column temperature, flow rate and delay

[0105] This study investigated key chromatographic parameters such as column temperature and flow rate to optimize separation. First, the effects of column temperatures of 30℃, 35℃, and 40℃ on resolution were examined, and the results are as follows: Figure 5 As shown, a column temperature of 35℃ resulted in optimal retention times for each chromatographic peak and the best separation effect. Next, the flow rates (0.9 mL / min, 1.0 mL / min, and 1.1 mL / min) were investigated. Figure 6 The results showed that at a flow rate of 1.0 mL / min, both peak resolution and analysis time were ideal. Furthermore, to ensure complete elution of all components within the analysis time, a delay test was performed, and chromatograms showing retention times twice that of the mobile phase were recorded (e.g., [image of chromatogram]). Figure 7As shown in the figure, no delayed peaks appeared within the set analysis time of 83 minutes. Based on the above results, the column temperature of 35℃ and the flow rate of 1.0 mL / min were finally determined as the chromatographic conditions for this method.

[0106] 3. Optimization of the preparation method of the test solution

[0107] 3.1 Examination of Extraction Methods

[0108] Take samples from the same batch (1531613), grind them finely, take about 2g, weigh them accurately, add 20mL of methanol accurately, weigh them, sonicate them (power 250W, frequency 40kHz) and heat them under reflux for 30 minutes, cool them, weigh them again, make up the weight loss with methanol, shake well, filter, take the filtrate, and analyze it according to the above chromatographic conditions.

[0109] Depend on Figure 8 The results show that there is no significant difference between ultrasound and reflux extraction in terms of the peak area of ​​each characteristic peak. For the sake of simplicity, ultrasound was chosen as the extraction method (the ultrasound treatment conditions mentioned in the whole text are consistent with those of this experiment, namely, power 250W and frequency 40kHz).

[0110] 3.2 Investigation of extraction solvent

[0111] Take samples from the same batch (1531613), grind them finely, take about 2g, weigh accurately, and add 20mL each of methanol, 70% methanol, 50% methanol, 30% methanol, and water. Sonicate for 30 minutes, prepare the test solution according to the above method, and determine the results.

[0112] Depend on Figure 9 The results show that when 70% methanol is used as the solvent, all characteristic peaks can be detected and the peak areas are relatively high, which takes into account the extraction efficiency of different polar components. Therefore, 70% methanol was chosen as the extraction solvent.

[0113] 3.3 Examination of extraction time

[0114] Take a sample from the same batch number (1531613), grind it finely, take about 2g, weigh it accurately, add 20mL of 70% methanol accurately, and sonicate it for 30, 60 and 90 minutes respectively. Prepare the test solution according to the above method and determine it.

[0115] Depend on Figure 10 The results show that there is no significant difference in the peak area of ​​each feature peak compared with 60 and 90 minutes of extraction. To save time, the extraction time of 30 minutes was selected.

[0116] 3.4 Investigation of the amount of extraction solvent

[0117] Take samples from the same batch (1531613), grind them finely, take about 2g, weigh accurately, and add 15mL, 20mL, and 50mL of 70% methanol respectively. Sonicate for 30 minutes, prepare the test solution according to the above method and determine it.

[0118] Depend on Figure 11 The results show that the response value is moderate when using 20 mL of solvent, so 20 mL of 70% methanol was selected as the extraction solvent.

[0119] In summary, the preparation method of the test solution is as follows: Take an appropriate amount of this product, grind it into a fine powder, take about 2g, weigh it accurately, place it in a stoppered conical flask, accurately add 20mL of 70% methanol solution, weigh it, sonicate it (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol solution, shake it well, filter it, and take the filtrate to obtain the test solution.

[0120] 4. Preparation of reference solution

[0121] Take appropriate amounts of crotonin reference standard, naringin reference standard, and magnolol reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 0.03 mg of crotonin, 0.3 mg of naringin, and 0.3 mg of magnolol per 1 mL, which is used as the reference solution.

[0122] 5. Establishment of a characteristic atlas of Gastrointestinal Comfort Pills

[0123] Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, and determine the chromatograms under the following chromatographic conditions. Chromatogram of the reference solution (…) Figure 12 In the chromatogram of the test sample solution, the chromatographic peaks of crotonin, naringin, and magnolol were well separated; Figure 13 In the chromatogram, the characteristic peaks are symmetrical, and the resolution meets the requirements. Simultaneously, a 70% methanol solution was used as a blank solvent, and the same method was used for determination. The chromatogram was recorded. Figure 14 The results showed that no chromatographic peaks appeared at the retention times corresponding to each characteristic peak in the blank solvent, indicating that the solvent did not interfere with the determination.

[0124] Chromatographic conditions:

[0125] Column: OSAKA SODA CAPCELL PAK C18 MGⅡ (250mm×4.6mm, 5μm);

[0126] Mobile phase: Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the system six program.

[0127] Detection wavelength: Wavelength switching technology is used, 290nm for 0-15 minutes and 225nm for 15-83 minutes;

[0128] Column temperature: 35℃; flow rate: 1.0 mL / min; theoretical plate number: calculated based on the naringin peak should be no less than 100,000.

[0129] Sixteen batches of Weichangan Pills samples (batch numbers: 1531492, 1531502, 1531515, 1531522, 1531533, 1531543, 1531556, 1531567, 1531575, 1531584, 1531592, 1531606, 1531614, 1531619, 1531622, 1531613) were collected. Test solutions were prepared according to the above method, and the results were measured. Chromatograms were recorded within 83 minutes. Figure 15 The obtained chromatograms were imported into the "Similarity Evaluation System for Chromatographic Characteristic Chromatograms of Traditional Chinese Medicine" (2012 edition). The median method was used to generate a reference characteristic chromatogram, and a total of 19 common characteristic peaks were identified. The results are shown below. Figure 16 .

[0130] Example 2: Characteristic chromatographic peak identification and medicinal flavor attribution analysis

[0131] To further clarify the origins of the common peaks in the characteristic chromatogram of Weichangan Pill, chromatographic peak identification and herbal attribute analysis were performed. Reference herbs including croton oil, artificial musk (provided by the manufacturer), jujube, chuanxiong rhizome, magnolia bark, rhubarb, agarwood, bitter orange peel, sandalwood, and costus root were prepared into individual herbal solutions according to the formulation process. Simultaneously, reference standards including crotonin, naringin, magnolol, ferulic acid, agaricol, rutin, hesperidin, neohesperidin, emodin, chrysophanol, costus root lactone, and dehydrocostus root lactone were prepared into reference standard solutions with appropriate solvents. Negative sample solutions lacking any of the herbs in the prescription were also prepared.

[0132] Take the above solution, inject it according to the planned chromatographic conditions, and record the chromatograms. Figures 16-28 The chromatograms of the test sample solution were compared with those of the reference medicinal material solutions, reference standard solutions, and negative sample solutions. The results were analyzed by comparing retention times and spectra. Figure 1 Consistency analysis confirmed the attribution of the 19 common peaks. Figures 16-28The results showed that each characteristic peak was clearly assigned, and the negative sample was not interfered with at the corresponding position. The identification and assignment results of the common peaks are as follows: Peak 1: consistent with the retention time of crotonin reference standard, from croton oil, designated as S1 peak; Peaks 2-3: from jujube; Peaks 4 and 12: from chuanxiong; Peak 5: from agarwood; Peak 6: from chuanxiong; Peak 7: from bitter orange peel; Peak 8: from sandalwood; Peak 9: consistent with the retention time of naringin reference standard, from bitter orange peel, designated as S2 peak; Peak 10: from bitter orange peel (hesperidin); Peak 11: from bitter orange peel (neohesperidin); Peaks 13 and 19: from rhubarb (peak 13 is emodin, peak 19 is chrysophanol); Peak 14: consistent with the retention time of magnolol reference standard, from magnolia bark, designated as S3 peak; Peaks 15-16: from costus root (peak 15 is costus lactone, peak 16 is dehydrocostus lactone); Peak 18: from magnolia bark (magnoliol).

[0133] The above results indicate that the feature map constructed in this invention can comprehensively reflect the chemical composition information of the ten medicinal materials in the prescription.

[0134] Example 3: Validation of Feature Mapping Methodology

[0135] 1. Repeatability test

[0136] Six samples of the same batch number (1531613) of Weichangan Pills were taken, and test solutions were prepared according to the method in Example 1. The samples were then injected and analyzed under the optimal chromatographic conditions of Example 1. The chromatograms are shown below. Figure 29 As shown. Using S1 peak (crotonin), S2 peak (naringin), and S3 peak (and magnolol) as references, the relative retention times of each characteristic peak and the corresponding S peak were calculated.

[0137] Table 1. Results of Relative Retention Time Tests in Repeatability Experiments (n=6)

[0138]

[0139] Table 2. Results of Relative Peak Area Tests in Repeatability Experiments (n=6)

[0140]

[0141] Results of repeatability tests (see Table 1-2) Figure 29The results show that the relative retention time RSD of each characteristic peak and its corresponding S-peak is less than 2.0%, and the relative peak area RSD is also less than 2.0%, all within ±10% of the specified values ​​(specified values ​​for relative retention time with S1 peak: peak 2 is 1.26, peak 3 is 1.63; specified values ​​for relative retention time with S2 peak: peak 4 is 0.54, peak 5 is 0.78, peak 6 is 0.84, peak 7 is 0.96, peak 8 is 0.97, peak 10 is 1.03, peak 11 is 1.07; specified values ​​for relative retention time with S3 peak: peak 12 is 0.93, peak 13 is 0.99, peak 15 is 1.01, peak 16 is 1.03, peak 17 is 1.04, peak 18 is 1.05, peak 19 is 1.09), indicating that the method has good repeatability.

[0142] 2. Precision test

[0143] Take the test solution of the same batch number (1531613) of Weichangan Pills, and inject it continuously 6 times under the optimal chromatographic conditions in Example 1, and record the chromatograms. Figure 30 Using S1 peak (crotonin), S2 peak (naringin), and S3 peak (and magnolol) as references, the relative retention times of each common peak were calculated.

[0144] Table 3. Precision test results of relative retention time (n=6)

[0145]

[0146] Table 4. Precision test results of relative peak area (n=6)

[0147]

[0148] From Tables 3 and 4 Figure 30 It can be seen that the RSD of the relative retention time of each common peak is less than 0.05%, and the RSD of the relative peak area is less than 2.2%. Moreover, the retention times of each characteristic peak in the chromatograms obtained from the six determinations are basically consistent, and the peak shapes overlap well, indicating that the instrument has good precision and meets the methodological requirements.

[0149] 3. Stability test

[0150] Take the test solution of Weichangan Pills (1531613), inject it at 0, 6, 12, 18, and 24 hours, and record the chromatograms. Figure 31 ). Calculate the relative retention times of each common peak and the S peak.

[0151] From Tables 5 and 6 Figure 31It can be seen that the RSD of the relative retention time of each common peak is less than 2.0%, the RSD of the relative peak area is less than 2.0%, and the similarity between the chromatograms measured at each time point and the chromatogram at 0 hours is greater than 0.99. This indicates that the test solution has good stability within 24 hours at room temperature and can meet the determination requirements of this method.

[0152] Table 5. Results of relative retention time in stability tests (n=6)

[0153]

[0154] Table 6. Results of relative peak area tests for stability (n=6)

[0155]

[0156] 4. Durability test

[0157] Take the same batch of Weichangan Pills (1531613) test solution, and use three different batches of OSAKASODA CAPCELL PAK C18 MGⅡ columns (batch numbers: A4AD 24377, A4AD 02474, A4AD 50811) for injection and determination under the optimal chromatographic conditions of Example 1. Record the chromatograms. Figure 32 ), and calculate the theoretical plate number of the naringin peak.

[0158] Depend on Figure 32 It can be seen that the chromatograms obtained from the three columns of different batches are basically consistent, with good separation of each characteristic peak and no significant difference in relative retention times, all within ±10% of the specified values, indicating that the method has good robustness to different batches of columns. The theoretical plate numbers calculated based on the naringin peak for the three columns are 272,169, 273,562, and 281,268, respectively, all far exceeding 100,000. To ensure the applicability of the method, this method specifies that the theoretical plate number calculated based on the naringin peak should not be less than 100,000.

[0159] Example 4: Application of the characteristic spectrum of Gastrointestinal Comfort Pills in quality testing

[0160] Take the sample of the gastrointestinal analgesia pills to be tested, prepare the test solution according to the method in Example 1, and perform the determination, and record the chromatogram.

[0161] Chromatographic system suitability test: The theoretical plate number, calculated based on the naringin peak, should be no less than 100,000 (if the column efficiency decreases due to column aging or performance differences, the system suitability requirements can be adjusted appropriately to ensure effective separation of each characteristic peak from adjacent peaks).

[0162] Sample quality judgment criteria:

[0163] (1) The chromatogram of the sample to be tested should show 19 characteristic peaks;

[0164] (2) The relative retention time of each characteristic peak and the corresponding S peak should be within ±10% of the specified value;

[0165] If the sample to be tested meets both of the above requirements, it is judged to be of qualified quality; otherwise, it is judged to be of unqualified quality.

[0166] This method was used to test 16 batches of commercially available gastrointestinal health pills. The results showed that all 16 batches exhibited 19 characteristic peaks, and the relative retention times of each peak were within ±10% of the specified values. Furthermore, the system suitability met the requirements (theoretical plate number not less than 100,000), indicating good quality uniformity of the commercially available products. This method can be used for the routine quality control and evaluation of gastrointestinal health pills.

[0167] Example 5: Evaluation of the similarity of feature maps of different batches of samples

[0168] Sixteen batches of normal samples of Weichangan Pills (batch numbers: 1531492, 1531502, 1531515, 1531522, 1531533, 1531543, 1531556, 1531567, 1531575, 1531584, 1531592, 1531606, 1531613, 1531614, 1531619, 1531622) and ten batches of laboratory-prepared taste-deficient negative samples were taken. Test solutions were prepared according to the method in Example 1 and measured. The obtained chromatograms were imported into the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine" (2012 edition), using the characteristic chromatogram constructed in Example 1 (…). Figure 16 Using the median method as a reference, with a time window width of 0.1 min, full-spectrum matching was performed to calculate the similarity.

[0169] Table 7. Similarity evaluation results of feature maps of samples from different batches.

[0170]

[0171] As shown in Table 7, the similarity of all 16 batches of normal samples was above 0.985 (minimum 0.985), while the similarity of the taste-deficient negative samples was below 0.85, indicating that this method can effectively distinguish between complete prescriptions and taste-deficient samples. Taking into account factors such as differences in the source of medicinal materials and fluctuations in production processes, and referring to the "Guidelines for the Application of the Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine," the similarity between the characteristic chromatogram of this product and the control characteristic chromatogram is tentatively set at no less than 0.90.

[0172] Comparative Example

[0173] To verify the specific detection capability of the method of the present invention for the non-volatile highly polar components of Weichangan Pills, samples from the same batch (batch number: 1531613) were compared and measured using the characteristic chromatographic method of the present invention, the GC-MS volatile characteristic chromatographic method, and the pharmacopoeia content determination method. Specifically, the method of the present invention prepared the test solution according to Example 1 and performed the determination; the GC-MS method followed the method of Example 1 in patent CN113984917B, taking an appropriate amount of sample, placing it in a headspace vial, adding water, sealing, and then performing headspace sampling; the pharmacopoeia content determination method followed the method under Weichangan Pills in Part I of the 2025 edition of the Chinese Pharmacopoeia, determining the content of magnolol and honokiol after extraction with methanol.

[0174] As shown in Table 8, the method of this invention can detect 19 non-volatile characteristic peaks, covering various types of chemical components such as crotonin, flavonoid glycosides (naringin, hesperidin, neohesperidin), anthraquinones (emodin, chrysophanol), and phenolic acids (ferulic acid), comprehensively covering the nine medicinal materials in the prescription. The GC-MS method mainly detects volatile terpenoid components and has no response to highly polar non-volatile components; the pharmacopoeia content determination method only detects two indicator components in Magnolia officinalis and cannot reflect the dosage of other medicinal materials. The method of this invention constructs a complete quality control system for Weichangan Pills, consisting of "volatile component characteristic spectrum + non-volatile component characteristic spectrum + multi-index content determination".

[0175] Table 8 Comparison of the detection capabilities of the method of the present invention and existing technologies

[0176]

Claims

1. A method for constructing a characteristic map of a gastrointestinal soothing pill, characterized in that, Includes the following steps: (1) Preparation of the test solution; (2) Preparation of the reference solution; (3) The reference solution and the test solution were determined by high performance liquid chromatography, and characteristic chromatograms were constructed. The chromatographic conditions are as follows: Octadecylsilane-bonded silica gel is used as a filler; Using acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, gradient elution was performed according to the following procedure: 0–10 minutes, mobile phase A 3% → 10%, mobile phase B (%) 97 → 90; Within 10–42 minutes, mobile phase A changed from 10% to 26%, and mobile phase B changed from 90% to 74%. 42–83 minutes, mobile phase A 26%→76%, mobile phase B (%) 74→24; Detection was performed using wavelength switching technology; the detection wavelength was 290nm for 0–15 minutes and 225nm for 15–83 minutes. The column temperature was 35℃; the flow rate was 1.0 mL / min.

2. The construction method according to claim 1, characterized in that, The reference solution mentioned in step (2) is a mixed solution containing crotonin, naringin and magnolol.

3. The construction method according to claim 1, characterized in that, The preparation method of the test solution in step (1) is as follows: take the sample of Weichangan pills, grind it into a fine powder, add 20 mL of 70% methanol solution, sonicate for 30 minutes, filter, and take the filtrate to obtain the solution.

4. The construction method according to claim 3, characterized in that, The ultrasonic extraction in step (1) uses a power of 250W and a frequency of 40kHz.

5. The construction method according to claim 1, characterized in that, The high-performance liquid chromatography method used an OSAKA SODA CAPCELL PAK C18 MGⅡ column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

6. A characteristic map of a gastrointestinal analgesic pill constructed using the method described in any one of claims 1 to 5, characterized in that, The characteristic spectrum has 19 common characteristic peaks, of which peak 1 is the S1 peak corresponding to the crotonin reference peak, peak 9 is the S2 peak corresponding to the naringin reference peak, and peak 14 is the S3 peak corresponding to the magnolol reference peak.

7. The feature map according to claim 6, characterized in that, The relative retention times of each characteristic peak and the S peak are within ±10% of the specified value; The specified relative retention times with peak S1 are: 1.26 for peak 2 and 1.63 for peak 3; The specified relative retention times with peak S2 are as follows: peak 4 is 0.54, peak 5 is 0.78, peak 6 is 0.84, peak 7 is 0.96, peak 8 is 0.97, peak 10 is 1.03, and peak 11 is 1.

07. The specified relative retention times with peak S3 are as follows: 0.93 for peak 12, 0.99 for peak 13, 1.01 for peak 15, 1.03 for peak 16, 1.04 for peak 17, 1.05 for peak 18, and 1.09 for peak 19.

8. The feature map according to claim 6, characterized in that, The medicinal materials with the common characteristic peaks are classified as follows: Peak 1 comes from croton oil, peaks 2-3 come from jujube, peaks 4 and 12 come from Sichuan lovage rhizome, peak 5 comes from agarwood, peak 6 comes from Sichuan lovage rhizome, peak 7 comes from bitter orange peel, peak 8 comes from sandalwood, peaks 9-11 come from bitter orange peel, peaks 13 and 19 come from rhubarb, peaks 14, 17 and 18 come from magnolia bark, and peaks 15-16 come from costus root.

9. The application of the characteristic chromatogram of the gastrointestinal analgesic pill according to any one of claims 6 to 8 in the quality detection of the gastrointestinal analgesic pill, characterized in that, Includes the following steps: (1) Construct a characteristic spectrum of the gastrointestinal analgesic pill sample to be tested according to the construction method of any one of claims 1 to 5; (2) Compare the characteristic spectrum of the sample to be tested with the characteristic spectrum of the gastrointestinal analgesia pills according to any one of claims 6 to 8 to determine the quality of the sample to be tested.

10. The application according to claim 9, characterized in that, The standard for judging the quality of the sample to be tested is as follows: (1) The chromatogram of the sample to be tested should show 19 characteristic peaks; (2) The relative retention time of each characteristic peak and the S peak should be within ±10% of the specified value, as described in claim 7; (3) Import the chromatogram of the sample to be tested into the similarity evaluation system of chromatographic feature chromatogram of traditional Chinese medicine, and calculate the similarity with the feature chromatogram of Weichangan Pill as described in claim 6. The similarity should not be less than 0.90.

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