Specific chromatogram construction method and identification method of irkutsk anemone rhizome medicinal material or preparation thereof

Abstract

The invention provides a specific chromatogram construction method of an irkutsk anemone rhizome medicinal material or a preparation thereof. The invention also provides a specific chromatogram construction method of irkutsk anemone rhizome decoction pieces, standard decoction and formula granules, and a method for identifying irkutsk anemone rhizome medicinal material adulterants and identifying rhizoma acori graminei. According to the specific chromatogram construction method, the problem that in the prior art, the technological process from the rhizoma anemones altaicae medicinal material to the rhizoma anemones altaicae formula granule finished product is difficult to integrally evaluate and control can be solved, quality detection on the rhizoma anemones altaicae is comprehensive and reliable in production, and large-scale application and popularization are facilitated; the method can also be used for detecting adulterants of rhizoma anemones altaicae medicinal materials and identifying rhizoma acori graminei medicinal materials or preparations, and is wide in application range.

Description

Technical Field

[0001] The invention relates to a method for constructing a characteristic spectrum of a Chinese calamus herb or a preparation thereof. Background Art

[0002] Acorus calamus is the dried rhizome of Anemone altaica Fisch.ex CAMey, a plant in the Ranunculaceae family. It has a pungent and warm nature and enters the Heart and Stomach meridians. It has the effects of invigorating the orifices, resolving phlegm, invigorating the spleen and calming the mind. It is used to treat coma due to febrile illness, epilepsy, tinnitus, deafness, chest tightness, abdominal distension, loss of appetite, and externally treat carbuncles, sores, and ringworms.

[0003] Currently, there are numerous misidentified varieties of nine-segmented calamus in the medicinal material market. These products primarily originate from plants of the same family (Ranunculaceae) or different genera and are easily misused due to similar appearances or similar names. Common misidentifications include Acorustatarinowii, Anemone amurensis, Anemone raddeana, Cardamine salviae, and Acorus calamus. For example, although not a member of the Ranunculaceae family, Acorustatarinowii is often mistaken for nine-segmented calamus due to its densely packed nodes but fibrous cross-section and a strong aromatic aroma. Authentic nine-segmented calamus, on the other hand, has dense, oblique nodes, circular vascular bundles in cross-section, and a slightly sour flavor. Distinguishing genuine products from misidentified varieties becomes even more difficult after the medicinal material has been processed and extracted.

[0004] The Zhejiang Province Formula Granule Quality Standard discloses the standard for Acorus calamus formula granules, using high-performance liquid chromatography to establish a characteristic spectrum. Compared to the present application, this standard presents fewer peaks (five) and identifies only one peak, adenosine. Furthermore, this standard does not specify relative peak areas, making it impossible to distinguish Acorus calamus from its impurities. Summary of the Invention

[0005] The present invention provides a characteristic spectrum construction method and an identification method for a Chinese calamus herb or a preparation thereof.

[0006] The present invention provides a method for constructing a characteristic spectrum of a Chinese calamus herb or a preparation thereof, comprising the following steps:

[0007] a. Prepare the test solution: Take the test sample raw material, dissolve it in a solvent, extract it, and obtain the test solution;

[0008] b. High performance liquid chromatography was used to determine the characteristic spectrum of the Acorus calamus medicinal material or its preparation, wherein the chromatographic conditions were as follows: a C18 column; methanol as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, gradient elution, and the gradient elution conditions were as follows:

[0009]

[0010] The method also includes preparing a reference solution: taking a uridine reference substance and an adenosine reference substance, adding water to dissolve them, and obtaining a reference solution.

[0011] Wherein, each 1 ml of the reference solution contains 10 μg of uridine and 2 μg of adenosine.

[0012] Wherein, the extraction method described in step a is ultrasonic extraction, and the ultrasonic extraction conditions are: power 600W, frequency 40kHz, ultrasonic time 20-40min; the amount of nine-section calamus and water is 25-50ml of water for every 0.5g.

[0013] Preferably, the ultrasonic time is 30 minutes, and the amount of Acorus calamus and water is 25 ml of water for every 0.5 g of Acorus calamus.

[0014] The chromatographic column described in step b is a C18 column with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the column temperature is 20-30°C, the flow rate is 0.6-1.0 ml per minute, the column temperature is 20-30°C, the detection wavelength is 254-285 nm, and the theoretical plate number calculated based on the uridine peak should be no less than 8000.

[0015] Preferably, the column temperature in step b is 25° C., the flow rate is 0.8 ml per minute, and the detection wavelength is 254 nm.

[0016] The characteristic spectrum contains 7 characteristic peaks, and the relative retention times are: peak 1: 0.25, peak 2: 0.34, peak 3: 1.0, peak 4: 1.11, peak 5: 1.24, peak 6: 1.68, peak 7: 2.03, and the relative retention times fluctuate within the range of ±10%; among them, peak 3 is uridine and peak 4 is adenosine.

[0017] The present invention also provides a method for constructing characteristic maps of Acorus calamus slices, standard decoctions, and formula granules, which adopts the method for constructing characteristic maps of Acorus calamus medicinal materials or their preparations.

[0018] The present invention also provides a method for identifying adulterated calamus medicinal materials, which is characterized by:

[0019] a. Weigh the sample to be identified and construct a characteristic spectrum according to the construction method described above;

[0020] b. Compare the characteristic spectra. If the relative retention times are consistent, it is Acorus calamus.

[0021] Preferably, in the characteristic spectrum comparison in step b, the relative peak area of ​​peak 5 and peak 3 shall not be less than 1.0; the relative peak area of ​​peak 6 and peak 3 shall not be less than 1.7.

[0022] Among them, the mixed product of the nine-section calamus medicinal material includes Acorus tatarinowii, Anemone amurensis, Anemone raddeana, Cardamine purse, and Acoruscalamus; among them, the mixed product of the nine-section calamus medicinal material is Acorus tatarinowii, and the peak areas of peak 4 and peak 5 of the nine-section calamus medicinal material and Acorus tatarinowii are different. The peak areas of the nine-section calamus medicinal material at peak 6 and peak 7 are greater than the peak area of ​​Acorus tatarinowii.

[0023] The present invention also provides a method for identifying Acorus calamus medicinal materials or preparations, which comprises the following steps:

[0024] a. Weigh the sample to be identified and construct a characteristic spectrum according to the construction method described above;

[0025] b. Comparison of characteristic spectra: if the relative retention times are: Peak 1: 0.23, Peak 2: 0.33, Peak 3: 1.0, Peak 4:

[0026] Peak 1: 1.10, Peak 5: 1.26, Peak 6: 1.70, Peak 7: 2.09, there is a characteristic peak at a relative retention time of 0.97; there is a characteristic peak at a relative retention time of 1.36, a total of 9 characteristic peaks; if the relative retention time fluctuates within the range of ±10%, it is Acorus tatarinowii medicinal material or preparation.

[0027] The object of the present invention is to provide a method for constructing a characteristic spectrum of the medicinal material of Acorus calamus and its decoction pieces, standard decoctions, and formula granules, which can solve the problem in the prior art that it is difficult to comprehensively evaluate and control the process from the medicinal material of Acorus calamus to the finished product of the Acorus calamus formula granules. The quality detection of Acorus calamus during production is comprehensive and reliable, which is convenient for large-scale promotion and application. The characteristic spectrum established by the present invention can also be used for the detection of adulterated products of Acorus calamus medicinal material and the identification of Acorus calamus medicinal material or preparation, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Reference characteristic spectrum (peak 3 (S): uridine; peak 4: adenosine; chromatographic column: HSS T3,

[0029] 4.6mm×250mm, 5μm);

[0030] Figure 2 Chromatograms of Jiujie Changpu Granules with different mobile phases;

[0031] Figure 3 Chromatograms of Jiujie Changpu Granules at different wavelengths (wavelengths from bottom to top are 254nm, 285nm, and 270nm);

[0032] Figure 4 Column temperature investigation;

[0033] Figure 5 Flow rate investigation;

[0034] Figure 6 Delayed chromatogram;

[0035] Figure 7 Investigation of extraction solvents;

[0036] Figure 8 Investigation of extraction methods;

[0037] Figure 9 Extraction time investigation;

[0038] Figure 10 Investigation of solvent dosage;

[0039] Figure 11 Identification of chromatographic peaks in the characteristic spectrum of Jiujie Changcalamus formula granules;

[0040] Figure 12 Uridine spectrum - reference;

[0041] Figure 13 Uridine spectrum - granules;

[0042] Figure 14 Adenosine spectrum - reference;

[0043] Figure 15 Adenosine Spectrum - Granules;

[0044] Figure 16 Surveys using different instruments;

[0045] Figure 17 Different chromatographic columns (Note: Each spectrum represents batch from bottom to top HSS T3, ZORBAXEclipse Plus C18, YMC-Triart C18);

[0046] Figure 18 Characteristic spectrum of Nine-section Acorus calamus formula granules-KL-JJCP-01;

[0047] Figure 19 Characteristic spectrum of Nine-section Acorus calamus formula granules-KL-JJCP-02;

[0048] Figure 20 Characteristic spectrum of Nine-section Acorus calamus formula granules-KL-JJCP-03;

[0049] Figure 21 Characteristic spectra of Nine-section Acorus Gramineus Formula Granules (Note: The spectra from bottom to top represent batches of Nine-section Acorus Gramineus Formula Granules KL-JJCP-01, KL-JJCP-02, and KL-JJCP-03);

[0050] Figure 22 Comparative spectrum of Jiujie Changcalamus Formula Granules (wherein, Peak 3 (S): uridine; Peak 4: adenosine; chromatographic column: HSS T3, 4.6mm×250mm, 5μm);

[0051] Figure 23 Characteristic spectrum of Acorus tatarinowii slices (peak 3 (S): uridine; peak 4: adenosine);

[0052] Figure 24 Comparison of characteristic spectra of Acorus tatarinowii and Acorus calamus slices (peak 3 (S): uridine; peak 4: adenosine). DETAILED DESCRIPTION

[0053] Example 1 Method for constructing characteristic spectrum of Acorus calamus medicinal material or its preparation of the present invention

[0054] [Characteristic spectrum] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0512).

[0055] Chromatographic conditions and system suitability testing were performed using octadecylsilane bonded silica gel as the filler (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); methanol as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate, 0.8 ml / min; column temperature, 25°C; detection wavelength, 254 nm. The number of theoretical plates, calculated based on the uridine peak, should be no less than 8,000.

[0056]

[0057]

[0058] Preparation of Reference Solution: 2g of Acorus calamus reference material was added to 25ml of water and ultrasonically treated (power 600W, frequency 40kHz) for 30 minutes. The mixture was cooled, shaken, and filtered. The filtrate was used as the reference material solution. Separately, an appropriate amount of uridine reference substance and adenosine reference substance were accurately weighed and added to water to prepare a mixed solution containing 10μg of uridine and 2μg of adenosine per 1ml. This was used as the reference material solution.

[0059] Preparation of test solution: Medicinal materials and decoction pieces: Take 2 g of Acorus calamus medicinal materials and decoction piece powder (passed through No. 3 sieve), place in a stoppered conical flask, add 25 ml of water, and ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate.

[0060] Standard decoction: Take 0.5g of the powder of Nine-section Acorus calamus decoction, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of water, stopper it tightly, weigh it, and treat it ultrasonically (power 600W, frequency 40kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate.

[0061] Formula granules: Take an appropriate amount of Acorus calamus granules, grind them into powder, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of water, stopper, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate.

[0062] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0063] The test sample chromatogram should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the reference medicinal material chromatogram. Peak 3 and Peak 4 should correspond to the retention times of the corresponding reference material peaks, respectively. The peak corresponding to the uridine reference material peak is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.34 (peak 2), 1.24 (peak 5), 1.68 (peak 6), 2.03 (peak 7). See Figure 1 .

[0064] Example 2 Characteristic Spectrum Construction Method of Acorus calamus Medicinal Material or Its Preparation of the Present Invention Detection Condition Selection Test

[0065] 1 Establishment of the characteristic spectrum of Jiujie Changcalamus formula granules

[0066] 1.1 Experimental instruments and materials

[0067] High performance liquid chromatograph: Waters e2695 high performance liquid chromatograph, Shimadzu LC-20AD high performance liquid chromatograph, Thermo Fisher high performance liquid chromatograph;

[0068] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instrument Co., Ltd.);

[0069] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0070] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0071] Chromatographic column: HSS T3 4.6*250mm, 5μm, Agilent 5TC(2)-C18 4.6*250mm, 5μm, Kromasil 100-5-C18 4.6*250mm, 5μm.

[0072] Methanol (Sigma-Aldrich, chromatographic grade); phosphoric acid (Chengdu Kelong Chemical Co., Ltd., chromatographic grade); water was ultrapure water; other reagents were of analytical grade.

[0073] Uridine (China Food and Drug Inspection Institute, batch number: 110887-202305, content 99.6%);

[0074] Adenosine (China Food and Drug Inspection Institute, batch number: 110879-202204, content 99.4%);

[0075] Acorus calamus control medicinal material (Chengdu Desite Biotechnology Co., Ltd., batch number: DSTYJ002901);

[0076] Acorus calamus (Batch numbers: YC-JJCP-01, YC-JJCP-02, YC-JJCP-03, YC-JJCP-04, YC-JJCP-05, YC-JJCP-06, YC-JJCP-07, YC-JJCP-08, YC-JJCP-09, YC-JJCP-10, YC-JJCP-11, YC-JJCP-12, YC-JJCP-13, YC-JJCP-14, YC-JJCP-15, YC-JJCP-161)

[0077] Nine-section calamus slices (batch number: YP-JJCP-01, YP-JJCP-02, YP-JJCP-03, YP-JJCP-04, YP-JJCP-05, YP-JJCP-06, YP-JJCP-07, YP-JJCP-08, YP-JJCP-09, YP-JJCP-10, YP-JJCP-11, YP-JJCP-12, YP-JJCP-13, YP-JJCP-14, YP-JJCP-15, YP-JJCP-16)

[0078] Standardized decoction of Acorus calamus (Batch numbers: BT-JJCP-01, BT-JJCP-02, BT-JJCP-03, BT-JJCP-04, BT-JJCP-05, BT-JJCP-06, BT-JJCP-07, BT-JJCP-08, BT-JJCP-09, BT-JJCP-10, BT-JJCP-11, BT-JJCP-12, BT-JJCP-13, BT-JJCP-14, BT-JJCP-15, BT-JJCP-16)

[0079] Acorus calamus formula granules (Batch numbers: KL-JJCP-01, KL-JJCP-02, KL-JJCP-03).

[0080] 1.2 Chromatographic conditions

[0081] Use octadecylsilane bonded silica gel as the packing (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); use methanol as mobile phase A and 0.4% phosphoric acid solution as mobile phase B, using gradient elution as specified in the table below; flow rate: 0.8 ml / min; column temperature: 25°C; detection wavelength: 254 nm. The number of theoretical plates, calculated based on the uridine peak, should be no less than 8000.

[0082]

[0083] 1.3 Preparation of reference solution

[0084] Take 2g of Acorus calamus reference medicinal material, add 25ml of water, and sonicate (power 600W, frequency 40kHz) for 30 minutes. Let cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Separately, take appropriate amounts of uridine reference substance and adenosine reference substance, accurately weigh them, and add water to make a mixed solution containing 10μg of uridine and 2μg of adenosine per 1ml. This will be the reference substance solution.

[0085] 1.4 Preparation of test solution

[0086] Take an appropriate amount of Acorus calamus granules, grind them into powder, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of water, stopper, weigh, ultrasonically treat (power 600W, frequency 40kHz) for 30 minutes, let cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain the product.

[0087] 1.5 Determination method

[0088] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0089] 1.6 Chromatographic conditions and system suitability test

[0090] 1.6.1 Mobile phase selection

[0091] Based on the experimental conditions proposed above, methanol-0.4% phosphoric acid, acetonitrile-0.4% acetic acid, and acetonitrile-0.4% formic acid were investigated respectively. The results are shown in Figure 2 .

[0092] The results showed that when the mobile phase was methanol-0.4% phosphoric acid, the chromatographic peak information content was larger, the separation was better, and the peak elution time was more appropriate. The mobile phase was tentatively determined to be acetonitrile-0.4% phosphoric acid.

[0093] 1.6.2 Wavelength Selection

[0094] Based on the experimental conditions proposed above, the diode array detector was used to scan the entire wavelength range of the test solution, and the chromatograms of the test solution at wavelengths of 254 nm, 270 nm, and 285 nm were extracted respectively. Figure 3 .

[0095] The results showed that the chromatographic peak information content was larger and the chromatogram baseline was more stable when the detection wavelength was 254 nm, so the detection wavelength was determined to be 254 nm.

[0096] 1.6.3 Column temperature investigation

[0097] Based on the experimental conditions proposed above, the column temperatures of 20℃, 25℃ and 30℃ were investigated. Figure 4 , Tables 1-4.

[0098] Table 1 Column temperature investigation-retention time

[0099]

[0100] Table 2 Column temperature investigation-relative retention time

[0101]

[0102] Table 3 Column temperature investigation-peak area

[0103]

[0104] Table 4 Column temperature investigation-relative peak area

[0105]

[0106]

[0107] The results showed that when the column temperature was 25℃, the chromatogram peaks were relatively symmetrical, the separation was good, the peak elution time was moderate, and the peaks were relatively complete. Therefore, the column temperature was tentatively set at 25℃.

[0108] 1.6.4 Flow rate investigation

[0109] Based on the experimental conditions proposed above, the flow rates of 0.6ml / min, 0.8ml / min and 1.0ml / min were investigated. Figure 5 , Table 5-8.

[0110] Table 5 Flow rate investigation-retention time

[0111]

[0112] Table 6 Flow rate investigation-relative retention time

[0113]

[0114] Table 7 Flow rate investigation-peak area

[0115]

[0116] Table 8 Flow rate investigation-relative peak area

[0117]

[0118]

[0119] The results showed that when the flow rate was 0.8 ml / min, the chromatogram peak shape was better and the separation was moderate. Therefore, the flow rate was determined to be 0.8 ml / min.

[0120] 1.6.5 Delay Experiment

[0121] Based on the experimental conditions proposed above, the chromatogram acquisition time was extended to 90 minutes. Figure 6 .

[0122] The results showed that the sample had basically no chromatographic peak after 45 minutes, so the sample detection time was set at 45 minutes.

[0123] In summary, the chromatographic conditions and system suitability testing for the characteristic spectrum of Nine-section Acorus Gramineus Formula Granules were determined as follows: octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below; flow rate 0.8 ml / min; column temperature 25°C; detection wavelength 254 nm. The number of theoretical plates calculated based on the uridine peak should be no less than 8000.

[0124]

[0125] 1.7 Preparation of test solution

[0126] 1.7.1 Extraction solvent investigation

[0127] Take 0.5g of Nine-section Acorus Graminae (batch number: KL-JJCP-01), grind it into powder, and use 25ml of each of 30% methanol, 50% methanol, 70% methanol, ethanol, methanol, and water as the extraction solvent for the test sample. Ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake well, filter, and take the filtrate to obtain the product. Figure 7 .

[0128] The results showed that when the extraction solvent was water, the peak shapes of the characteristic peaks were good and the separation was moderate, so the extraction solvent was determined to be water.

[0129] 1.7.2 Extraction method investigation

[0130] Take 0.5g of Nine-section Acorus Graminae (batch number: KL-JJCP-01), grind it into powder, add 25ml of water, and examine the extraction method of the test sample when it is refluxed and ultrasonic (power 600W, frequency 40kHz), the extraction time is 30 minutes, let it cool, shake well, filter, and take the filtrate. Figure 8 .

[0131] The results showed that there was little difference in the effects of ultrasonic and reflux extraction of the test sample. The peak shapes and separation of each characteristic peak were good, and the ultrasonic method was fast and simple. Therefore, ultrasonic extraction was determined to be the extraction method for the test sample.

[0132] 1.7.3 Extraction time investigation

[0133] Take 0.5g of Nine-section Acorus Gramineus Granules (batch number: KL-JJCP-01), grind it into powder, add 25ml of water, and treat it with ultrasound (power 600W, frequency 40kHz). The extraction time of the test sample was 20 minutes, 30 minutes, and 40 minutes respectively. Let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 9 .

[0134] The results showed that the extraction was sufficient when the extraction time was 30 minutes, so the extraction time was determined to be 30 minutes.

[0135] 1.7.4 Investigation of solvent addition amount

[0136] Take 0.5g of Nine-section Acorus Graminae (batch number: KL-JJCP-01), grind it into powder, add 20ml, 25ml and 50ml of water respectively, treat it with ultrasound (power 600W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, and take the filtrate to obtain the product. Figure 10 .

[0137] The results showed that when the amount of solvent added was 25 ml, the peak shape and separation of each chromatographic peak were better, so the amount of solvent added was selected as 25 ml.

[0138] In summary, the preparation method of the test solution of the characteristic spectrum of the Jiujie Acorus calamus formula granules is determined as follows: take 0.5 g of Jiujie Acorus calamus granules, place it in a stoppered conical flask, add 25 ml of water, weigh it, ultrasonicate it for 30 minutes, let it cool, weigh it again, make up the weight with water, and filter it.

[0139] 1.8 Methodological Review

[0140] 1.8.1 Chromatographic peak identification

[0141] Preparation of test solution: According to the experimental conditions proposed above, prepare the test solution of Acorus calamus formula granules.

[0142] Preparation of reference solution: Take 2 g of Acorus calamus reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, weigh it, sonicate for 30 minutes, let it cool, weigh it again, make up the weight with water, filter it, and take the filtrate as the reference medicinal material solution.

[0143] Take an appropriate amount of uridine reference substance, weigh it accurately, and add aqueous solution to make a solution containing 10ug of uridine per 1ml.

[0144] Take an appropriate amount of adenosine reference substance, weigh it accurately, and add water to make a solution containing 2 μg of adenosine per 1 ml, which serves as the reference substance solution.

[0145] Preparation of negative control solution: According to the experimental conditions proposed above, prepare the negative control solution of Acorus calamus formula granules.

[0146] The characteristic spectrum peaks of the Jiujie Changpu formula granules were located. Figure 11-14 .

[0147] The results showed that peak 4 was uridine and peak 5 was adenosine. In the following methodological investigation, eight characteristic peaks in the sample were investigated.

[0148] 1.8.2 Precision test

[0149] Take the test solution of the Acorus calamus formula granules and inject 10 μl of the solution six times according to the proposed experimental method. Calculate the relative retention time and relative peak area of ​​each characteristic peak. See Tables 9-10.

[0150] Table 9 Precision Investigation-Relative Retention Time

[0151]

[0152] Table 10 Precision investigation-relative peak area

[0153]

[0154] The results showed that the RSD range of the relative retention times of the characteristic peaks of the samples was 0.00-0.22%, indicating that the instrument had good precision.

[0155] 1.8.3 Repeatability Study

[0156] Accurately weigh 6 portions of Acorus calamus granules and prepare and measure them according to the proposed experimental method. See Tables 11-12.

[0157] Table 11 Repeatability study - relative retention time

[0158]

[0159] Table 12 Repeatability study - relative peak area

[0160]

[0161] The results showed that the RSD of the relative retention time of the six samples ranged from 0.00% to 0.17%, indicating that the method had good repeatability.

[0162] 1.8.4 Intermediate precision study

[0163] 1.8.4.1 Inspection of different instruments

[0164] Based on the experimental conditions proposed above, the Nine-section Acorus Gramineus Formula Granules (Batch No.: KL-JJCP-01) were accurately weighed to prepare the test solution, which was then measured on Waters, Thermo Fisher, and Shimadzu high performance liquid chromatographs. Figure 16 , Tables 13-14.

[0165] Table 13 Instrument durability investigation - relative retention time

[0166]

[0167] Table 14 Instrument durability investigation - relative peak area

[0168]

[0169] The results showed that when the three instruments were used to detect the test samples, the RSDs of the relative retention times of the characteristic peaks were 0.41% to 6.55%, and the durability of the instruments was good.

[0170] 1.8.4.2 Inspection by different personnel and time

[0171] Based on the experimental conditions proposed above, different individuals (A and B) accurately weighed Acorus calamus formula granules (Batch No.: KL-JJCP-01) at different times (T1 and T2) to prepare test samples for measurement. See Tables 15-16.

[0172] Table 15 Personnel and time inspection - relative retention time

[0173]

[0174] Table 16 Personnel and time investigation-relative peak area

[0175]

[0176]

[0177] The results showed that when different people measured the same sample at different times, the RSD of the relative retention time of each characteristic peak was 0.00% to 0.22%, indicating that the method was stable.

[0178] 1.8.5 Column Durability Assessment

[0179] Based on the experimental conditions proposed above, three HSS T3 4.6*250mm, 5μm (chromatographic column 1), Agilent 5TC(2)-C18 4.6*250mm, 5μm (chromatographic column 2), Kromasil 100-5-C18 4.6*250mm, 5μm (chromatographic column 3) were used for the analysis. Figure 17 , Tables 17-18.

[0180] Table 17 Column durability investigation - relative retention time

[0181]

[0182] Table 18 Column durability inspection - relative peak area

[0183]

[0184] The results showed that when the samples were detected using the three chromatographic columns, the RSDs of the relative retention times of the characteristic peaks were between 0.79% and 10.40%, and all three chromatographic columns were capable of detecting the characteristic peaks.

[0185] 1.8.6 Stability Study

[0186] Based on the experimental conditions proposed above, take the same test solution and measure it at 0h, 2h, 6h, 9h, 15h, and 24h respectively. See Table 19-20.

[0187] Table 19 Stability Study-Relative Retention Time

[0188]

[0189] Table 20 Stability Study-Relative Peak Area

[0190]

[0191] The results showed that the RSDs of the characteristic peaks relative to the retention times were between 0.00% and 0.43%, and the sample solutions were stable within 24 hours.

[0192] In summary, although Peak 3 has poor peak shape and low resolution, the RSDs of the relative retention times of all characteristic peaks except Peak 3 meet the requirements in all the above investigations, indicating that this method is good. The seven characteristic peaks except Peak 3 will be included in the subsequent investigation.

[0193] 1.8.7 Determination of characteristic peaks and establishment of reference maps

[0194] 1.8.7.1 Verification Results of Three Batches of Acorus calamus Formula Granules

[0195] The proposed method was used to determine the characteristic spectra of three batches of Jiujie Changpu formula granules, and the relative retention time and relative peak area were calculated. Figure 18-21 , Tables 21-22.

[0196] Table 21 Relative retention time of Acorus calamus formula granules

[0197]

[0198]

[0199] Table 22 Relative peak area of ​​Acorus calamus formula granules

[0200]

[0201] Seven peaks with good reproducibility were selected as characteristic peaks based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights. Based on the methodological review results and validation results from three batches of particles, the theoretical plate number, calculated based on the uridine peak, was tentatively set to be no less than 10,000.

[0202] 1.8.7.2 Establishment of Relative Retention Time Limits

[0203] The summary of the methodology inspection items and verification results is shown in Table 23:

[0204] Table 23 Summary of RSD% results for each item of the methodology - relative retention time

[0205]

[0206] The relative retention time of each characteristic peak is stable and within the range of ±10% of the average value, so the specified value range of the relative retention time of each peak is temporarily set at ±10%; the relative peak area of ​​each batch varies too much to be specified, so the relative peak area is not included in the text.

[0207] Final regulations: The test sample chromatogram should show seven characteristic peaks, and their retention times should correspond to those of the seven characteristic peaks in the chromatogram of the reference medicinal material. Peaks 3 and 4 should correspond to the retention times of the corresponding reference material peaks, respectively. The peak corresponding to the uridine reference material peak is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.25 (peak 1), 0.34 (peak 2), 1.24 (peak 5), 1.68 (peak 6), and 2.03 (peak 7).

[0208] Three batches of Jiujie Changpu formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison spectrum of the characteristic spectrum of Jiujie Changpu formula granules was established. Figure 22 .

[0209] Conclusion: The characteristic spectrum method of Jiujie Changcalamus formula granules can be used to effectively detect Jiujie Changcalamus formula granules.

[0210] In summary, this method is suitable for the detection of high-performance liquid chromatography characteristic patterns of Acorus calamus medicinal materials, decoction pieces, standard decoctions, and formula granules. It can comprehensively control the characteristic components in Acorus calamus medicinal materials, decoction pieces, standard decoctions, and formula granules, ensuring the overall stability of the quality of Acorus calamus medicinal materials, decoction pieces, standard decoctions, and formula granules. The method is simple to operate, has high precision, good stability, good repeatability, and high accuracy.

[0211] Example 3 Identification Method of Confused Products of Acorus calamus

[0212] The proposed method was used to determine the characteristic spectra of three batches of Acorus tatarinowii slices. Figure 23 , Tables 24-25.

[0213] Table 24 Relative retention time of Acorus tatarinowii slices

[0214]

[0215] Table 25 Relative peak area of ​​Acorus tatarinowii slices

[0216]

[0217] Three batches of Acorus tatarinowii slices were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version). A reference spectrum of the characteristic spectrum of Acorus tatarinowii slices was established and compared with the reference spectrum of Acorus tatarinowii slices. Figure 24 .

[0218] The results showed that: 1. At the relative retention time of 0.97, Acorus calamus had one more characteristic peak than Acorus tatarinowii; 2. The peak areas at peaks 4 and 5 of Acorus tatarinowii were significantly different from those of Acorus calamus; 3. The peak areas at peaks 6 and 7 of Acorus tatarinowii were much larger than those of Acorus calamus; 4. At the relative retention time of 1.36, Acorus calamus had one more peak than Acorus tatarinowii.

[0219] 3. Summary of identification points

[0220] By comparing the characteristic spectra of the medicinal materials, decoction pieces, standard decoctions and formula granules of Jiujie Acorus calamus with the medicinal materials, decoction pieces, standard decoctions and formula granules of Shijiao Acorus tatarinowii, the results showed that: 1. At the relative retention time of 0.97, Shijiao Acorus tatarinowii has one more characteristic peak than Jiujie Acorus calamus; 2. The peak areas at peaks 4 and 5 in Jiujie Acorus calamus are significantly different from the peak areas at these same points of Shijiao Acorus tatarinowii; 3. The peak areas at peaks 6 and 7 of Jiujie Acorus calamus are much larger than the peak areas at these same points of Shijiao Acorus tatarinowii; 4. At the relative retention time of 1.36, Shijiao Acorus tatarinowii has one more peak than Jiujie Acorus calamus.

[0221] By comparing the relative peak areas of the two and summarizing them, see Table 26:

[0222] Table 26 Summary of relative peak areas of Acorus calamus and Acorus tatarinowii

[0223]

[0224]

[0225] The results showed that the relative peak areas of Peak 5 / Peak 3 and Peak 6 / Peak 3 were significantly different between the medicinal materials, decoction pieces, standard decoctions, and formula granules of Acorus tatarinowii and those of Acorus tatarinowii. The maximum peak areas of Peak 5 / Peak 3 and Peak 6 / Peak 3 in Acorus tatarinowii were smaller than the minimum values ​​in Acorus tatarinowii. Therefore, the relative peak area ratios of Peak 5 / Peak 3 and Peak 6 / Peak 3 can be used as a distinguishing criterion between Acorus tatarinowii and the standard decoction of Acorus tatarinowii.

[0226] By comparing the characteristic spectra of the medicinal materials, decoction pieces, standard decoctions and formula granules of Jiujie Acorus calamus with the medicinal materials, decoction pieces, standard decoctions and formula granules of Shijiao Acorus tatarinowii, the results showed that: 1. At the relative retention time of 0.97, the medicinal materials, decoction pieces, standard decoctions and formula granules of Shijiao Acorus tatarinowii had one more characteristic peak than that of Jiujie Acorus calamus; 2. The peak areas at peaks 4 and 5 in the medicinal materials, decoction pieces, standard decoctions and formula granules of Jiujie Acorus calamus were significantly different from the peak areas at these locations of Shijiao Acorus tatarinowii; 3. The peak areas at peaks 6 and 7 in the medicinal materials, decoction pieces, standard decoctions and formula granules of Jiujie Acorus calamus were much larger than the peak areas at these locations of Shijiao Acorus tatarinowii; 4. At the relative retention time of 1.36, the medicinal materials, decoction pieces, standard decoctions and formula granules of Shijiao Acorus tatarinowii had one more peak than that of Jiujie Acorus calamus; 5. Calculate Peak 5 and Peak 3. The relative peak area of ​​Peak 6 and Peak 3 should be within the specified range, which is: not less than 1.0 (peak 5) Not less than 1.7 (peak 6) .

[0227] This characteristic method can quickly and effectively identify Acorus calamus and its mixed and counterfeit products, and the method is simple to operate, high in precision, good in stability, good in repeatability and high in accuracy.

Claims

1. A method for constructing a characteristic spectrum of a Chinese calamus herb or its preparation, characterized in that: It includes the following steps: a. Prepare the test solution: Take the test sample raw material, dissolve it in a solvent, extract it, and obtain the test solution; b. High performance liquid chromatography was used to determine the characteristic spectrum of the Acorus calamus medicinal material or its preparation, wherein the chromatographic conditions were as follows: a C18 column; methanol as mobile phase A, 0.4% phosphoric acid solution as mobile phase B, gradient elution, and the gradient elution conditions were as follows:

2. The method for constructing a characteristic spectrum of the Acorus calamus medicinal material or its preparation according to claim 1, characterized in that: The method also includes preparing a reference solution: taking a uridine reference substance and an adenosine reference substance, adding water to dissolve them, and obtaining a reference solution; each 1 ml of the reference solution contains 10 μg of uridine and 2 μg of adenosine.

3. The method for constructing a characteristic spectrum of the medicinal material Acorus calamus or its preparation according to claim 1, characterized in that: The extraction method in step a is ultrasonic extraction, and the ultrasonic extraction conditions are: power 600W, frequency 40kHz, and ultrasonic time 20-40min; the amount of Acorus calamus and water is 25-50ml of water for every 0.5g; Preferably, the ultrasonic time is 30 minutes, and the amount of Acorus calamus and water is 25 ml of water for every 0.5 g of Acorus calamus.

4. The method for constructing a characteristic spectrum of the Acorus calamus medicinal material or its preparation according to claim 1, characterized in that: The chromatographic column described in step b is a C18 column with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the column temperature is 20-30°C, the flow rate is 0.6-1.0 ml per minute; the column temperature is 20-30°C; the detection wavelength is 254-285 nm; the theoretical plate number calculated based on the uridine peak should be not less than 8000; preferably, the column temperature in step b is 25°C, the flow rate is 0.8 ml per minute, and the detection wavelength is 254 nm.

5. The method for constructing a characteristic spectrum of the Acorus calamus medicinal material or its preparation according to any one of claims 1 to 4, characterized in that: The characteristic spectrum contains 7 characteristic peaks, and the relative retention times are: peak 1: 0.25, peak 2: 0.34, peak 3: 1.0, peak 4: 1.11, peak 5: 1.24, peak 6: 1.68, peak 7: 2.03, and the relative retention times fluctuate within the range of ±10%; among them, peak 3 is uridine and peak 4 is adenosine.

6. A method for constructing characteristic maps of Acorus calamus slices, standard decoctions, and formula granules, characterized by: The method adopts the characteristic spectrum construction method of the Acorus calamus medicinal material or its preparation according to any one of claims 1 to 5.

7. A method for identifying adulterated Acorus calamus medicinal materials, characterized by: a. Weigh the sample to be identified and construct a characteristic spectrum according to the construction method according to any one of claims 1 to 6; b. Compare the characteristic spectra. If the relative retention times are consistent, it is Acorus calamus.

8. The method for identifying adulterated products of Acorus calamus according to claim 7, wherein: In the characteristic spectrum comparison in step b, the relative peak area between Peak 5 and Peak 3 shall not be less than 1.0; the relative peak area between Peak 6 and Peak 3 shall not be less than 1.

7.

9. The method for identifying adulterated products of Acorus calamus according to claim 7 or 8, characterized in that: The mixed product of the Acorus tatarinowii medicinal material includes Acorus tatarinowii, Anemone amurensis, Anemone raddeana, Cardamine purse, and Acorus calamus; among them, the mixed product of the Acorus tatarinowii medicinal material is Acorus tatarinowii, the peak areas of the Acorus tatarinowii medicinal material and Acorus calamus at peaks 4 and 5 are different, and the peak areas of the Acorus tatarinowii medicinal material at peaks 6 and 7 are greater than the peak area of ​​Acorus calamus.

10. A method for identifying Acorus tatarinowii medicinal materials or preparations, characterized by: It includes the following steps: a. Weigh the sample to be identified and construct a characteristic spectrum according to the construction method according to any one of claims 1 to 6; b. Comparison of characteristic spectra: if the relative retention time is: Peak 1: 0.23, Peak 2: 0.33, Peak 3: 1.0, Peak 4: Peak 1: 1.10, Peak 5: 1.26, Peak 6: 1.70, Peak 7: 2.09, there is a characteristic peak at a relative retention time of 0.97; there is a characteristic peak at a relative retention time of 1.36, a total of 9 characteristic peaks; if the relative retention time fluctuates within the range of ±10%, it is Acorus tatarinowii medicinal material or preparation.

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