A fingerprint quality control method for safflower compositions

Abstract

The application discloses a fingerprint spectrum construction method of a Huahong composition, and comprises gradient elution and determination by a high performance liquid chromatograph; the high performance liquid chromatograph conditions are as follows: a chromatographic column is a reversed-phase C18 chromatographic column; a detection wavelength is 326 nm; and a mobile phase is that a mobile phase A is 0.1% formic acid aqueous solution, and a mobile phase B is methanol, and gradient elution is adopted. The UPLC fingerprint spectrum of the Huahong composition is established by optimizing chromatographic conditions and establishing a test product preparation methodology. The method is simple, accurate, and good in repeatability, and can better control the quality consistency of the Huahong composition.

Description

Technical Field

[0001] This invention relates to the field of quality control of traditional Chinese medicine, specifically to a fingerprint chromatogram quality control method for a safflower composition. Background Technology

[0002] The Huahong composition, represented by Huahong tablets, consists of the following medicinal ingredients: *Hedyotis diffusa*, *Hedyotis diffusa*, *Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gnaphalium affine*, and *Iris tectorum*. Xiaozhong Zhitong Ding is a traditional Chinese medicine product of Guangxi Zhuang Autonomous Region Huahong Pharmaceutical Group Co., Ltd. It clears heat and detoxifies, dries dampness and stops leukorrhea, removes blood stasis and relieves pain. It is used for leukorrhea and irregular menstruation caused by damp-heat stagnation, characterized by excessive, yellow, thick leukorrhea, lower abdominal pain, lumbosacral pain, and menstrual cramps; it is also used for chronic pelvic inflammatory disease and adnexitis with the above symptoms.

[0003] Fingerprinting technology is globally recognized as the most effective means of controlling the quality of complex systems, and it plays a significant role in the component determination and authenticity identification of traditional Chinese medicine or natural drugs. It has been gradually recognized that quality control methods targeting only a single chemical component can no longer adequately reflect the intrinsic quality of a product. An effective way to solve this problem is to establish innovative quality control methods and characteristic fingerprinting control models for standard preparations, thereby strengthening the scientific and standardized evaluation of traditional Chinese medicine quality. Therefore, constructing fingerprinting profiles for safflower compositions is of great importance. Summary of the Invention

[0004] The purpose of this invention is to provide a fingerprint chromatographic quality control method for safflower compositions. This invention establishes a UPLC fingerprint chromatogram for safflower compositions through chromatographic condition optimization and sample preparation methodology. This method is simple, accurate, and has good repeatability, enabling effective control of the quality consistency of safflower compositions.

[0005] The technical content of this invention is as follows:

[0006] The fingerprint chromatographic quality control method for safflower compositions includes gradient elution and determination using high performance liquid chromatography;

[0007] The high-performance liquid chromatography conditions are as follows:

[0008] Chromatographic column: Reversed-phase C18 column;

[0009] Detection wavelength: 326nm;

[0010] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is used, and the elution program is as follows:

[0011] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-18 90→76 10→24 18-23 76 24 23-60 76→50 24→50 .

[0012] The reversed-phase C18 column has the following specifications: 3.0×150mm, 3.5μm; the column temperature is 20-30℃; and the mobile phase flow rate is 0.4-1.0mL / min.

[0013] The reversed-phase C18 column is a water XTERRA@RP18 column.

[0014] The method for preparing the test solution includes the following steps:

[0015] Take 0.5-2g of safflower tablets, safflower capsules, or safflower granules, grind them into a fine powder, add 4-8ml of 65% methanol solution, sonicate for 20-40min, cool, let stand, and filter to obtain the final product.

[0016] The method for preparing the reference solution includes the following steps:

[0017] Take neochlorogenic acid reference standard, add 65% methanol and mix, sonicate for 20-40 minutes, cool, and prepare to 0.1-0.3 mg / ml.

[0018] The standard fingerprint spectrum was established as follows: 5 μl of the reference solution and 15 batches of test solution were injected into the liquid chromatograph, measured, and the chromatograms were recorded. Neochlorogenic acid in the chromatogram of the test sample was identified as the reference peak. Detection was performed at 326 nm, and 14 characteristic peaks were obtained. The relative retention time and relative peak area of ​​each common peak were calculated to obtain the standard fingerprint spectrum formed by the 14 common peaks.

[0019] The characteristic peaks of the standard fingerprint spectrum are as follows:

[0020]

[0021] The aforementioned method for quality control of the fingerprint spectrum of safflower composition includes the following:

[0022] (1) Preparation of the test solution:

[0023] Take 0.5-2g of safflower tablets, safflower capsules, or safflower granules, grind them into a fine powder, add 4-8ml of 65% methanol solution, sonicate for 20-40min, cool, let stand, and filter to obtain the product.

[0024] (2) Preparation of reference solution:

[0025] Take neochlorogenic acid reference standard, add 65% methanol and mix, sonicate for 20-40 min, cool, and prepare to a concentration of 0.1-0.3 mg / ml.

[0026] (3) Chromatographic conditions:

[0027] Chromatographic column: reversed-phase C18 column, 3.0×150mm, 3.5μm; column temperature: 20-30℃;

[0028] Detection wavelength: 326nm;

[0029] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is used, with a mobile phase flow rate of 0.4-1.0 mL / min. The elution program is as follows:

[0030] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-18 90→76 10→24 18-23 76 24 23-60 76→50 24→50

[0031] (4) Establishment of standard fingerprint spectrum: Take 5 μl of reference solution and 15 batches of test solution, inject them into liquid chromatograph, measure and record the chromatogram, and determine the neochlorogenic acid in the chromatogram of the test sample as the reference peak; detect at 326 nm to obtain 14 characteristic peaks, calculate the relative retention time and relative peak area of ​​each common peak, and thus obtain the standard fingerprint spectrum formed by the 14 common peaks;

[0032] (5) Using the methods described in (1)-(3) as the means of testing the fingerprint spectrum of the safflower composition to be tested, prepare the fingerprint spectrum of the sample to be tested;

[0033] (6) The fingerprint spectrum of the red composition to be tested is compared with the standard fingerprint spectrum in step (4), and the similarity is greater than 0.9.

[0034] The aforementioned safflower composition is made from the following medicinal materials: *Hedyotis diffusa*, ...Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gardenia jasminoides*, and *Ipomoea quamoclit*.

[0035] The beneficial effects of this invention are:

[0036] This invention realizes a fingerprint spectrum quality control mode and quality consistency control method for safflower compositions, which can accurately control and evaluate the quality of safflower compositions and make up for the shortcomings of existing quality control methods.

[0037] The method of this invention is simple, accurate, and highly repeatable, and has good application prospects. Attached Figure Description

[0038] Figure 1 UPLC overlay spectra of 15 batches of safflower granule samples;

[0039] Figure 2 Chromatograms for precision and stability experiments;

[0040] Figure 3 Chromatograms for repeatability experiments;

[0041] Figure 4The total ion chromatogram of safflower particles in UPLC-MS / MS positive and negative ion modes;

[0042] Figure 5 UPLC fingerprint spectra of 15 batches of safflower granule samples;

[0043] Figure 6 The UPLC spectrum of the standard neochlorogenic acid is shown. Detailed Implementation

[0044] The present invention will be further illustrated below by way of examples. It should be understood that the examples of the present invention are for illustrative purposes and not for limiting the present invention.

[0045] Example 1

[0046] The quality control method for the fingerprint spectrum of safflower compositions includes the following:

[0047] (1) Preparation of the test solution:

[0048] Take 0.5g of safflower tablets, grind them into fine powder, add 4ml of 65% methanol solution, sonicate for 20min, cool, let stand, and filter to obtain the final product.

[0049] (2) Preparation of reference solution:

[0050] Take neochlorogenic acid reference standard, add 65% methanol, mix, sonicate for 20 min, cool, and prepare to 0.1 mg / ml.

[0051] (3) Chromatographic conditions:

[0052] Chromatographic column: water XTERRA@RP18 column, 3.0×150mm, 3.5μm; column temperature: 20℃;

[0053] Detection wavelength: 326nm;

[0054] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is used, with a mobile phase flow rate of 0.4-1.0 mL / min. The elution program is as follows:

[0055] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-18 90→76 10→24 18-23 76 24 23-60 76→50 24→50

[0056] (4) Establishment of standard fingerprint spectrum: Take 5 μl of reference solution and 15 batches of test solution, inject them into liquid chromatograph, measure and record the chromatogram, and determine the neochlorogenic acid in the chromatogram of the test sample as the reference peak; detect at 326 nm to obtain 14 characteristic peaks, calculate the relative retention time and relative peak area of ​​each common peak, and thus obtain the standard fingerprint spectrum formed by the 14 common peaks;

[0057] The characteristic peaks of the standard fingerprint spectrum are as follows:

[0058]

[0059]

[0060] (5) Using the methods described in (1)-(3) as the means of testing the fingerprint spectrum of the safflower composition to be tested, prepare the fingerprint spectrum of the sample to be tested;

[0061] (6) The fingerprint spectrum of the red composition to be tested is compared with the standard fingerprint spectrum in step (4), and the similarity is greater than 0.9.

[0062] Example 2

[0063] The quality control method for the fingerprint spectrum of safflower compositions includes the following:

[0064] (1) Preparation of the test solution:

[0065] Take 2g of Huahong capsule sample, grind it into fine powder, add 8ml of 65% methanol solution, sonicate for 40min, cool, let stand, filter, and the product is obtained.

[0066] (2) Preparation of reference solution:

[0067] Take neochlorogenic acid reference standard, add 65% methanol, mix, sonicate for 40 min, cool, and prepare to 0.3 mg / ml.

[0068] (3) Chromatographic conditions:

[0069] Chromatographic column: water XTERRA@RP18 column, 3.0×150mm, 3.5μm; column temperature: 30℃;

[0070] Detection wavelength: 326nm;

[0071] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is used, with a mobile phase flow rate of 1.0 mL / min. The elution program is as follows:

[0072] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-18 90→76 10→24 18-23 76 24 23-60 76→50 24→50

[0073] (4) Establishment of standard fingerprint spectrum: Take 5 μl of reference solution and 15 batches of test solution, inject them into liquid chromatograph, measure and record the chromatogram, and determine the neochlorogenic acid in the chromatogram of the test sample as the reference peak; detect at 326 nm to obtain 14 characteristic peaks, calculate the relative retention time and relative peak area of ​​each common peak, and thus obtain the standard fingerprint spectrum formed by the 14 common peaks;

[0074] The characteristic peaks of the standard fingerprint spectrum are as follows:

[0075]

[0076] (5) Using the methods described in (1)-(3) as the means of testing the fingerprint spectrum of the safflower composition to be tested, prepare the fingerprint spectrum of the sample to be tested;

[0077] (6) The fingerprint spectrum of the red composition to be tested is compared with the standard fingerprint spectrum in step (4), and the similarity is greater than 0.9.

[0078] Example 3

[0079] The quality control method for the fingerprint spectrum of safflower compositions includes the following:

[0080] (1) Preparation of the test solution:

[0081] Take 1.5g of safflower tablets, grind them into a fine powder, add 4-8ml of 65% methanol solution, sonicate for 25min, cool, let stand, and filter to obtain the product;

[0082] (2) Preparation of reference solution:

[0083] Take neochlorogenic acid reference standard, add 65% methanol, mix, sonicate for 33 min, cool, and prepare to 0.2 mg / ml.

[0084] (3) Chromatographic conditions:

[0085] Chromatographic column: water XTERRA@RP18 column, 3.0×150mm, 3.5μm; column temperature: 26℃;

[0086] Detection wavelength: 326nm;

[0087] Mobile phase: Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol. Gradient elution was used, with a mobile phase flow rate of 0.8 mL / min. The elution program was as follows:

[0088] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-18 90→76 10→24 18-23 76 24 23-60 76→50 24→50

[0089] (4) Establishment of standard fingerprint spectrum: Take 5 μl of reference solution and 15 batches of test solution, inject them into liquid chromatograph, measure and record the chromatogram, and determine the neochlorogenic acid in the chromatogram of the test sample as the reference peak; detect at 326 nm to obtain 14 characteristic peaks, calculate the relative retention time and relative peak area of ​​each common peak, and thus obtain the standard fingerprint spectrum formed by the 14 common peaks;

[0090] The characteristic peaks of the standard fingerprint spectrum are as follows:

[0091]

[0092]

[0093] (5) Using the methods described in (1)-(3) as the means of testing the fingerprint spectrum of the safflower composition to be tested, prepare the fingerprint spectrum of the sample to be tested;

[0094] (6) The fingerprint spectrum of the red composition to be tested is compared with the standard fingerprint spectrum in step (4), and the similarity is greater than 0.9.

[0095] Example 4

[0096] The quality control method for the fingerprint spectrum of safflower compositions includes the following:

[0097] (1) Preparation of the test solution:

[0098] Take 1g of cinnamon granules, grind them into fine powder, add 65% methanol solution to make up to 5ml, sonicate for 30min, add 65% methanol solution to make up to 5ml, cool, let stand, filter, and the product is obtained.

[0099] (2) Preparation of reference solution:

[0100] Take neochlorogenic acid reference standard, add 65% methanol, mix, sonicate for 30 min, cool, and prepare to 0.2 mg / ml.

[0101] (3) Chromatographic conditions:

[0102] Chromatographic column: water XTERRA@RP18 column, 3.0×150mm, 3.5μm; column temperature: 25℃;

[0103] Detection wavelength: 326nm;

[0104] Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is used, with a mobile phase flow rate of 0.4-1.0 mL / min. The elution program is as follows:

[0105] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-18 90→76 10→24 18-23 76 24 23-60 76→50 24→50

[0106] (4) Establishment of standard fingerprint spectrum: Take 5 μl of reference solution and 15 batches of test solution, inject them into liquid chromatograph, measure and record the chromatogram, and determine the neochlorogenic acid in the chromatogram of the test sample as the reference peak; detect at 326 nm to obtain 14 characteristic peaks, calculate the relative retention time and relative peak area of ​​each common peak, and thus obtain the standard fingerprint spectrum formed by the 14 common peaks;

[0107] The characteristic peaks of the standard fingerprint spectrum are as follows:

[0108]

[0109]

[0110] (5) Using the methods described in (1)-(3) as the means of testing the fingerprint spectrum of the safflower composition to be tested, prepare the fingerprint spectrum of the sample to be tested;

[0111] (6) The fingerprint spectrum of the red composition to be tested is compared with the standard fingerprint spectrum in step (4), and the similarity is greater than 0.9.

[0112] The aforementioned safflower composition is made from the following medicinal materials: *Hedyotis diffusa*, ...Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gardenia jasminoides*, and *Ipomoea quamoclit*.

[0113] Example 5: Establishment and Methodological Investigation of the Fingerprint Spectrum of Safflower Particles

[0114] 1. Experimental Materials

[0115] 1.1 Instruments

[0116] Agilent 1290 ultra-high performance liquid chromatograph; electronic balance, 1 / 100,000 (Sartorius Scientific Instruments Co., Ltd.); ultrasonic cleaner (Yumeng); liquid chromatography-mass spectrometry system (SHIMADZU Corporation, Japan);

[0117] 1.2 Medicines and Reagents

[0118] 15 batches of Huahong granules (Guangxi Huahong Pharmaceutical Co., Ltd.) (see Table 1); methanol (chromatographic grade, Merck); formic acid (analytical grade, Tianjin Fuyu Fine Chemical Co., Ltd.); distilled water (Watsons).

[0119]

[0120] 2 Experimental Methods

[0121] 2.1 Establishment of UPLC fingerprint spectrum

[0122] 2.1.1 Preparation of the test solution

[0123] Accurately weigh 1g of cinnamon granules powder, place it in a 5ml volumetric flask, add 65% methanol to the mark, mix well, sonicate for 30min, cool, make up the weight difference, let stand, and set aside for later use.

[0124] 2.1.2 Preparation of reference solution

[0125] Accurately weigh 1 mg of neochlorogenic acid and place it in a 5 ml volumetric flask. Add 65% methanol to the mark, mix well, sonicate for 30 min, cool, make up the weight difference, and let stand for later use.

[0126] 2.1.2 Chromatographic conditions

[0127] Chromatographic column: water XTERRA@RP18 (3.0×150mm, 3.5μm); mobile phase: 0.1% formic acid (A), methanol (B); gradient elution: (0-18min 90%A-76%A, 18-23min 76%A, 23-60min 76%A-50%A), detection wavelength: 326nm, column temperature: 25℃, flow rate: 0.6ml / min, injection volume: 5μl.

[0128] 2.2 Methodological Examination

[0129] 2.2.1 Precision Examination

[0130] Accurately weigh the S14 sample, prepare the cinnamon granule test solution according to the method in "2.1.1", inject it into the ultra-high performance liquid chromatograph, perform 6 parallel injections, record the peak area and retention time, and calculate the relative standard deviation (RSD).

[0131] 2.2.2 Repeatability Test

[0132] Accurately weigh 6 portions of S14 sample, prepare test solutions according to method "2.1.1", inject the samples separately, and calculate the relative retention time, relative peak area and relative standard deviation (RSD) of each chromatographic peak.

[0133] 2.2.3 Stability Test

[0134] Accurately weigh the S14 sample and prepare the test solution according to the method in "2.1.1". Inject the sample at 0, 2, 4, 8, 12 and 24 h respectively to examine its stability, and calculate the relative retention time, relative peak area and relative standard deviation (RSD) of each chromatographic peak.

[0135] 2.3 Identification of common peaks and similarity evaluation of fingerprint spectra

[0136] UPLC chromatographic data were imported into the National Pharmacopoeia Commission's "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version) in AIA format. Using the averaging method, a time window of 0.1 seconds was selected, and S4 was used as the reference spectrum. Multi-point correction and Mark matching were performed to establish overlay spectra for 15 batches of samples (see...). Figure 1 ) and comparison atlas (see Figure 6 The common peaks were identified, and the similarity of fingerprint spectra of 15 batches of samples was calculated.

[0137] 2.4 Analysis of Common Peaks in the Fingerprint Spectrum of Safflower Particles

[0138] 2.4.1 Mass Spectrometry Conditions

[0139] Mass spectrometry conditions: Electrospray ionization (ESI) source, positive and negative ion modes, scan range m / z 100–1000; collision energy 35

[0140] eV; ion transfer tube temperature 320℃; vaporizer temperature 350℃; positive and negative ion spray voltages are 3500 and 3000V respectively.

[0141] 2.4.2 Data Analysis

[0142] The analysis was performed using Compound Discoverer 3.0 software. Five databases were used for matching analysis: Predicted Compositions, mzCloudSearch, mzVaultSearch, ChemSpider Search, and MassList Search. Then, based on the ppm value range of [-5 to +5], the analysis was further filtered and matched.

[0143] 3 Results

[0144] 3.1 Identification of common peaks and similarity evaluation of fingerprint spectra

[0145] Establish overlay patterns of 15 batches of safflower granules (see...) Figure 1 ) and comparison atlas (see Figure 6 The results showed that there were 14 common peaks with similarity ranging from 0.977 to 1.000, indicating good similarity (Table 2).

[0146] Table 2. Similarity evaluation results of fingerprint spectra of 15 batches of safflower granules

[0147]

[0148] 3.2 Methodological Examination

[0149] 3.2.1 Precision and stability tests

[0150] Using peak 1 as a reference peak, the relative retention times and relative peak areas of all common peaks in the fingerprint spectrum were recorded. The relative retention time RSD was <0.7%, and the relative peak area RSD was <4.3%. This indicates good instrument precision and stability of the cinnamon granule test solution within 0-24 hours. The results are as follows... Figure 2 As shown in Tables 3 and 4 below.

[0151] Table 3. Relative peak areas of each peak in the fingerprint spectrum for precision and stability experiments (n=6)

[0152]

[0153] 1 2 3 4 5 6 1(S) 1.00 1.00 1.00 1.00 1.00 1.00 0.00％ 2 0.17 0.17 0.18 0.18 0.18 0.18 1.79％ 3 1.06 1.05 1.05 1.06 1.08 1.07 1.19％ 4 1.00 1.00 1.00 1.01 1.04 1.02 1.53％ 5 0.94 0.93 0.93 0.93 0.96 0.96 1.39％ 6 0.58 0.56 0.56 0.55 0.58 0.58 2.35％ 7 1.88 1.87 1.88 1.90 1.95 1.91 1.49％ 8 0.45 0.44 0.44 0.45 0.45 0.45 1.22％ 9 0.12 0.12 0.12 0.12 0.12 0.12 2.69％ 10 0.79 0.79 0.80 0.80 0.82 0.83 2.13％ 11 0.30 0.29 0.30 0.30 0.31 0.30 2.02％ 12 0.06 0.06 0.07 0.06 0.07 0.06 3.28％ 13 0.29 0.29 0.29 0.29 0.30 0.29 1.45％ 14 0.20 0.19 0.18 0.19 0.20 0.20 4.33％

[0154] Table 4. Relative retention times of each peak in the fingerprint chromatograms for precision and stability experiments (n=6)

[0155]

[0156] 3.2.2 Repeatability Test

[0157] Using peak 1 as a reference peak, the relative retention times and relative peak areas of all common peaks in the fingerprint spectrum were recorded. The relative retention time RSD < 0.47% and the relative peak area RSD < 2.6%, meeting the requirements for fingerprint spectra. This indicates good reproducibility of the experiment, and the results are as follows. Figure 3 As shown in Tables 5 and 6 below.

[0158] Table 5. Relative peak areas of each peak in the fingerprint spectrum of repeatability test (n=6)

[0159]

[0160] Table 6. Relative retention times of each peak in the fingerprint spectrum of the repeatability test (n=6)

[0161]

[0162]

[0163] 3.3 Analysis of common peaks in the fingerprint spectrum of cinnamon particles

[0164] 3.3.1 Qualitative Analysis of Common Peaks in the Fingerprint Spectrum of Safflower Granules

[0165] The pollen particles were analyzed using UPLC-MS / MS technology. The sample solution was scanned using both positive and negative ion modes. The total ion chromatogram is shown below. Figure 4 .according to Figure 1 The UPLC fingerprint spectra of 15 batches of Huahong granules were analyzed, and 14 common peaks were qualitatively analyzed. The results are shown in Table 6. Among them, the chemical composition of peak 1, which was used as the reference peak in the fingerprint analysis, is Neochlorogenic acid from Yidianhong, Jixueteng, Baihuasheshecao, Ximing, and Ditaohua.

[0166]

[0167]

[0168] 3.3.2 Identification of reference peaks and determination of content in the fingerprint spectrum of cinnamon granules

[0169] By comparing the chromatograms with those of the reference standard, the S peak was identified as Neochlorogenic acid. Figure 5 and Figure 6By performing a simple conversion between the concentration of chlorogenic acid in peak S and the concentration of chlorogenic acid in standard No. 1 (0.2 mg / mL), the concentration of chlorogenic acid in each batch of samples can be roughly calculated to be 33-38 ug / mL.

[0170] 4. Research Summary

[0171] The UPLC fingerprint spectrum of the Huahong granules obtained in this experiment contained 14 common peaks, which were identified by UPLC-MS / MS as Neochlorogenic acid, Esculin, Chlorogenic acid, Ferulic acid, Daidzein, Phloretin, Quercetin, etc. Among them, the S peak (peak 1) was identified as Neochlorogenic acid by comparison with the reference standard spectrum.

[0172] In addition, the similarity of all 15 batches of safflower granules in this experiment was greater than 0.97, indicating good similarity and suggesting that the chemical composition of each batch of commercially available safflower granules was relatively consistent.

[0173] This study established a UPLC fingerprint of cinnamon granules by optimizing chromatographic conditions and preparing the test sample. The method is simple, accurate, and reproducible.

[0174] The above are embodiments of the present invention. The above embodiments and specific parameters are only for clearly illustrating the invention verification process and are not intended to limit the patent protection scope of the present invention. The patent protection scope of the present invention shall still be determined by its claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of a safflower composition, characterized in that: This includes gradient elution and determination using high-performance liquid chromatography; The high performance liquid chromatography conditions are as follows: Chromatographic column: Reversed-phase C18 column; Detection wavelength: 326 nm; Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is used, and the elution program is as follows: The preparation method of the test solution includes the following steps: Take 0.5-2g of Huahong tablets, Huahong capsules, or Huahong granules, grind them into a fine powder, add 4-8ml of 65% methanol solution, sonicate for 20-40min, cool, let stand, and filter to obtain the product. The standard fingerprint spectrum was established as follows: 5 μl of the reference solution and 15 batches of test solution were injected into the liquid chromatograph, measured, and the chromatograms were recorded. Neochlorogenic acid in the chromatogram of the test sample was identified as the reference peak. Detection was performed at 326 nm, and 14 characteristic peaks were obtained. The relative retention time and relative peak area of ​​each common peak were calculated to obtain the standard fingerprint spectrum formed by the 14 common peaks. The aforementioned safflower composition is made from the following medicinal materials: *Hedyotis diffusa*, *Hedyotis diffusa*, *Spatholobus suberectus*, *Rhodomyrtus tomentosa* root, *Bai Bei Ye* root, *Gnaphalium affine*, and *Ipomoea aquatica*.

2. The method for constructing the fingerprint spectrum of the cinnamon composition as described in claim 1, characterized in that: The reversed-phase C18 column has the following specifications: 3.0 × 150 mm, 3.5 μm; the column temperature is 20-30℃; and the mobile phase flow rate is 0.4-1.0 mL / min.

3. The method for constructing the fingerprint spectrum of the cinnamon composition as described in claim 2, characterized in that: The reversed-phase C18 column is a water XTERRA@ RP18 column.

4. The method for constructing the fingerprint spectrum of the cinnamon composition as described in claim 1, characterized in that: The method for preparing the reference solution includes the following steps: Take neochlorogenic acid reference standard, add 65% methanol and mix, sonicate for 20-40 minutes, cool, and prepare to 0.1-0.3 mg / ml.

5. The method according to claim 1, wherein the method is characterized by, The method includes the following: (1) Preparation of the test solution: Take 0.5-2g of Huahong tablets, Huahong capsules, or Huahong granules, grind them into a fine powder, add 4-8ml of 65% methanol solution, sonicate for 20-40min, cool, let stand, and filter to obtain the product. (2) Preparation of reference solution: Take neochlorogenic acid reference standard, add 65% methanol, mix, sonicate for 20-40 min, cool, and prepare to a concentration of 0.1-0.3 mg / ml. (3) Chromatographic conditions: Chromatographic column: reversed-phase C18 column, 3.0 × 150 mm, 3.5 μm; column temperature: 20-30℃; Detection wavelength: 326 nm; Mobile phase: Mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is methanol. Gradient elution is used, with a mobile phase flow rate of 0.4-1.0 mL / min. The elution program is as follows: (4) Establishment of standard fingerprint: 5 μl of the control solution and 5 μl of the 15 batches of test solution were injected into the liquid chromatograph, and the chromatogram was recorded to determine the neochlorogenic acid in the test sample chromatogram as the reference peak; detection was performed at 326 nm to obtain 14 characteristic peaks, and the relative retention time and relative peak area of each common peak were calculated to obtain the standard fingerprint formed by the 14 common peaks; (5) The methods described in (1)-(3) were used as the testing means of the fingerprint of the test sample; (6) The fingerprint of the test sample was compared with the standard fingerprint of step (4), and the similarity was greater than 0.9.

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