UPLC (Ultra Performance Liquid Chromatography) quantitative fingerprint spectrum detection method for Cinnamomum red

Abstract

The invention provides a UPLC (Ultra Performance Liquid Chromatography) quantitative fingerprint spectrum detection method for pericarpium citri reticulatae, and belongs to the technical field of quality detection of traditional Chinese medicines. The detection method comprises the following steps: preparing a reference substance solution, preparing a test solution, analyzing by using ultra-high performance liquid chromatography, introducing multiple batches of liquid chromatograms into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, identifying 21 common peaks, and carrying out similarity matching on the 21 common peaks to generate a reference map of the cinnamomum camphora red pericarpium citri reticulatae. On the basis, the quantitative fingerprint spectrum of the cinnamomum camphora pericarpium citri reticulatae is established by combining the determination of the content of five flavonoid components, namely hesperidin, sweet orange flavone, nobiletin, tangeretin and 5-demethylnobiletin. According to the method disclosed by the invention, the fingerprint detection of the cinnamomum camphora red dried tangerine peel and the content determination of the five flavonoid components can be simultaneously completed through one-time analysis, the quality of the cinnamomum camphora red dried tangerine peel can be rapidly, comprehensively and accurately evaluated, good reproducibility and stability are realized, and a technical support can be provided for the uniformity and stability of the quality of the cinnamomum camphora red dried tangerine peel.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a UPLC quantitative fingerprint detection method for red tangerine peel of Camphor Tree. Background Art

[0002] Zhangtouhong C.reticulate'Zhangshuensis' is one of the cultivated varieties of dried tangerine peel. It is a good variety of natural hybrid of Jiangxi Zhoushang red orange and local sour orange. It is a local variety of dried tangerine peel in Zhangshu City, Jiangxi Province. Zhangtouhong dried tangerine peel was included in the list of "Ten Foods of Jiangxi" in 2020. It is a local medicinal material with both medicinal and edible properties. It can be used as medicine and as tea. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and has huge market prospects.

[0003] The chemical composition of traditional Chinese medicine is extremely complex, with diverse chemical structures and a wide range of polarity distribution. Traditional microscopic identification and physical and chemical analysis are difficult to characterize the quality of traditional Chinese medicine, and conventional qualitative and quantitative quality control of multiple components requires the development of multiple analytical methods, which is costly and difficult to implement. In recent years, there have been no reports on the UPLC fingerprint of red tangerine peel and the determination of the content of multiple flavonoid components. At present, there is no literature that publicly applies the quantitative fingerprint method to the quality control of red tangerine peel. A more systematic quality control method has not yet been established, which seriously restricts the industrial development of red tangerine peel. Summary of the invention

[0004] In view of this, the present invention aims to provide a UPLC quantitative fingerprint detection method for tangerine peel, aiming to solve the problems that the development of fingerprints and multi-index component quantification in the prior art usually requires the development of multiple analysis methods, resulting in high cost of the operation method and difficulty in promotion. Quantitative fingerprints use one analysis method to simultaneously realize fingerprints and multi-index component content determination, significantly improving detection efficiency and reducing detection costs.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a UPLC quantitative fingerprint detection method for red tangerine peel, which specifically comprises steps S11 to S15.

[0007] S11: Preparation of reference solution: Hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin were taken as reference substances, and 50-100% methanol (V / V) or 50-100% ethanol (V / V) were added to prepare mixed reference solution;

[0008] S12: Preparation of test solution: Take an appropriate amount of Zhangtouhong Chenpi sample, accurately weigh it, put it into a stoppered conical flask, add a methanol solvent with a volume concentration of 50% to 100% or an ethanol solvent with a volume concentration of 50% to 100%, and the solid-liquid ratio (ratio of sample to methanol solvent or ethanol solvent) is 1:25 to 1:500 g / mL (preferably 1:100 to 1:150 g / mL), ultrasonically or refluxed for 10 to 60 min, make up the lost weight with the solvent, shake well, centrifuge, and filter the supernatant to obtain the Zhangtouhong Chenpi test solution;

[0009] S13: Ultra-high performance liquid chromatography (UPLC) analysis: Prepare 3 or more (preferably 10 or more, more preferably 20 or more) different batches of camphor red tangerine peel test solution according to the method in step 2, respectively draw the reference solution and the test solution, respectively inject them into the ultra-high performance liquid chromatograph, respectively record the liquid chromatograms of the reference and the test, and record the peak areas of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in the reference and the test;

[0010] S14: Establishment of fingerprint: importing the liquid chromatograms of at least 3 (preferably more than 10) different batches of Zhangtouhong Chenpi test samples into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", matching the common peaks, obtaining a control fingerprint, identifying the common peaks, and establishing the Zhangtouhong Chenpi fingerprint;

[0011] S15: Establishment of content determination method: According to the peak area of ​​each component recorded in S13, the external standard method ( Among them A i represents the peak area of ​​the component to be measured in the sample, Rf represents the correction factor, and m represents the sample weight) Calculate the contents of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in the samples of Zhangtouhong tangerine peel;

[0012] Further, the concentrations of the mixed reference solution in step S11 are 200-300 μg hesperidin, 1-10 μg sweet orange flavonoids, 20-60 μg nobiletin, 10-40 μg tangeretin, and 1-10 μg 5-demethylnobiletin per 1 mL, respectively;

[0013] Further, the ultra-high performance liquid chromatography conditions in step S13 are: the chromatographic column is ZORBAX SB-C18 (2.1×100 mm, 1.8 μm), the column temperature is 30-40° C., the flow rate is 0.3-0.5 mL / min, the injection volume is 0.5-2 μL, the detection wavelength is 283 nm, 330 nm, acetonitrile is used as the mobile phase A, and formic acid with a volume percentage concentration of 0.1-1.0% is used as the mobile phase B for gradient elution, and the elution gradient conditions are: 0-2 min, 8% A; 2-10 min, 8%-18% A; 10-15 min, 18%-22% A; 15-20 min, 22%-37% A; 20-28 min, 37% A; 28-30 min, 37%-90% A.

[0014] Further, there are 21 common peaks in the control fingerprint of step S14, among which peak 1 is 3-O-caffeoylquinic acid, peak 2 is vicinin-2, peak 3 is stellarin-2, peak 4 is limonin 17-β-D-glucopyranoside, peak 5 is naringin, peak 6 is hesperidin, peak 7 is dicaffeoylquinic acid, peak 8 is nomilin17-β-D-glucopyranoside, peak 9 is dicaffeoylquinic acid methyl ester, peak 10 is fennel glycoside, peak 11 is natsudaidain-3-O-(5-α-glucosyl-HMG)-β-glucoside, Peak 12 is hydroxy-trimethoxyflavone, peak 13 is hydroxy-tetramethoxyflavone, peak 14 is sweet orange flavone, peak 15 is 6-demethoxy tangeretin, peak 16 is nobiletin, peak 17 is 4',5,6,7-tetramethoxy flavone, peak 18 is 3,3',4',5,6,7,8-heptamethoxy flavone, peak 19 is 5-hydroxy-6,7,3',4'-tetramethoxy flavone, peak 20 is tangeretin, peak 21 is 5-demethyl nobiletin;

[0015] Further, in the step S14, the chromatogram of the test solution of Camphor Red Tangerine Peel at a wavelength of 330nm should show 21 characteristic peaks, of which 5 peaks should correspond to the retention time of the reference peaks of the reference substance, and the similarity is calculated based on the 21 common peaks, and the similarity between the test sample chromatogram and the reference spectrum is not less than 0.95.

[0016] Further, in the step S15, the contents of sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in the test solution of Camphor Red Tangerine Peel are calculated by the external standard method using the peak area in the chromatogram at a detection wavelength of 330 nm, and the content of hesperidin is calculated by the external standard method using the peak area in the chromatogram at a detection wavelength of 283 nm.

[0017] Further, in the step S15, the hesperidin (C 28 H 34 O 15 ) shall not be less than 2.59%; containing sweet orange flavonoids (C 20 H 20 O7) shall not be less than 0.025%; containing nobiletin (C 21 H 22 O8) shall not be less than 0.35%; containing tangeretin (C 20 H 20 O7) shall not be less than 0.17%; 5-demethylnobiletin (C 20 H 20 O8) shall not be less than 0.043%.

[0018] In a specific embodiment of the present invention, 21 common peaks were identified and similarity matching was performed with the 21 common peaks to generate a control spectrum of the red tangerine peel of Camphor Head. Combined with the content determination of five flavonoid components, including hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin, a quantitative fingerprint spectrum of the red tangerine peel of Camphor Head was established.

[0019] The present invention can simultaneously realize the fingerprint spectrum detection and content determination of the red tangerine peel of Zhangtou through one analysis, can quickly, comprehensively and accurately evaluate the quality of the red tangerine peel of Zhangtou, and provide technical support for the uniform quality of the red tangerine peel of Zhangtou.

[0020] The present invention has the following advantages:

[0021] 1. The UPLC quantitative fingerprint detection method for red tangerine peel provided by the present invention can realize the fingerprint detection of red tangerine peel and the content determination of 5 flavonoid components through one analysis, and can quickly, comprehensively and accurately evaluate the quality of red tangerine peel, and provide technical support for the uniform quality of red tangerine peel;

[0022] 2. The UPLC quantitative fingerprint detection method for red tangerine peel provided by the present invention uses the Chinese medicine chromatographic fingerprint similarity evaluation system provided by the National Pharmacopoeia Committee to construct a reference fingerprint for the measured fingerprint and calculate the similarity. The operation is convenient and fast, and the conclusion is objective and accurate.

[0023] 3. The UPLC quantitative fingerprint detection method for the red tangerine peel provided by the present invention can effectively control the quality of the red tangerine peel by determining five flavonoid components, namely, hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin;

[0024] 4. The UPLC quantitative fingerprint detection method for red tangerine peel provided by the present invention has carried out methodological investigations on the precision, repeatability, stability, etc. of the fingerprint, and methodological investigations on the linearity, sample recovery rate, precision, repeatability, stability, etc. of the content determination. The method is accurate and reliable, providing a more comprehensive quality control method for red tangerine peel. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings used in the specific implementation manner or the description of the prior art will be briefly introduced below.

[0026] Figure 1 It is the fingerprint superposition diagram of 16 batches of Zhangtouhong Chenpi in Example 1;

[0027] Figure 2 The reference fingerprint and common peaks in Example 1;

[0028] Figure 3 The standard curves of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in Example 2;

[0029] Figure 4 It is a comparison chart of the mixed reference substance and the determination of the content of Zhangtouhong Chenpi in Example 2. DETAILED DESCRIPTION

[0030] The UPLC quantitative fingerprint detection method for red tangerine peel of the present invention is further described in detail below in conjunction with specific examples.

[0031] Example 1: Establishment of UPLC fingerprint method of Zhangtouhong Chenpi

[0032] 1.1 Instruments, reagents, test drugs and their sources

[0033] Instruments: Waters Acquity I Class ultra-high performance liquid chromatograph; Agilent Infinity II 1290-6545Q-TOF LC-MS Agilent ultra-high performance liquid chromatograph-quadrupole time-of-flight mass spectrometer; ML204T / 02 1 / 10,000 analytical balance; XSE105 1 / 100,000 analytical balance; KQ-500DV sonicator; Waters UPLC-DAD chromatographic conditions ZORBAXSB-C18 column (2.1×100 mm, 1.8 μm); SOrvall ST8 high-speed centrifuge;

[0034] Reagents: acetonitrile (Thermo) and formic acid (Thermo) were of mass spectrometry grade; formic acid (Macklin) and acetonitrile (Fisher) were of chromatography grade; methanol (Energy Chemical) was of analytical grade; ultrapure water;

[0035] Test drugs: Reference substances hesperidin (batch number 110721-202220, mass fraction 97.2%), nobiletin (batch number 112055-202102, mass fraction 99.7%), and tangeretin (batch number 112054-202102, mass fraction 99.7%) were purchased from China Food and Drug Inspection Institutes, 5-demethylnobiletin (batch number PS011601, mass fraction 98.5%) and sweet orange flavonoids (batch number PS011273, mass fraction 98.0%) were purchased from Pusi Biotechnology Co., Ltd.;

[0036] The test sample was identified as C. reticulate'Zhangshuensis'. Specific information is shown in Table 1.

[0037] Table 1 Information on samples of red tangerine peel

[0038] batch number Origin Name of medicinal material source Origin A2312001-1 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-2 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-3 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-4 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-5 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-6 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-7 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-8 Camphor red Tangerine peel Origin collection Tongjiang Village, Zhoushang Township, Zhangshu City, Yichun City, Jiangxi Province A2312001-9 Camphor red Tangerine peel Origin collection Sanhu Town, Xingan County, Ji'an City, Jiangxi Province A2312001-10 Camphor red Tangerine peel Origin collection Sanhu Town, Xingan County, Ji'an City, Jiangxi Province A2312001-11 Camphor red Tangerine peel Origin collection Sanhu Town, Xingan County, Ji'an City, Jiangxi Province A2312001-12 Camphor red Tangerine peel Origin collection Sanhu Town, Xingan County, Ji'an City, Jiangxi Province A2312001-13 Camphor red Tangerine peel Origin collection Sanhu Town, Xingan County, Ji'an City, Jiangxi Province A2312001-14 Camphor red Tangerine peel Origin collection Xingan County, Ji'an City, Jiangxi Province A2312001-15 Camphor red Tangerine peel Origin collection Xingan County, Ji'an City, Jiangxi Province A2312001-16 Camphor red Tangerine peel Origin collection Xingan County, Ji'an City, Jiangxi Province

[0039] 1.2 Solution preparation

[0040] 1.2.1 Preparation of reference solution

[0041] Preparation of reference mixed standard solution: Take appropriate amount of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin reference substances, accurately weigh them, and add methanol to make a mixed solution containing 234.1 μg of hesperidin, 4.9 μg of sweet orange flavonoids, 40.8 μg of nobiletin, 20.9 μg of tangeretin, and 4.9 μg of 5-demethylnobiletin per 1 mL.

[0042] 1.2.2 Preparation of test solution

[0043] Take about 0.2 g of red tangerine peel powder (passed through No. 2 sieve), weigh it accurately, put it in a stoppered conical flask, add 25 mL of methanol accurately, weigh the mass, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, cool, weigh the mass again, make up the lost mass with methanol, shake well, filter, and take the filtrate to obtain.

[0044] 1.3 Chromatographic conditions

[0045] Chromatographic column: ZORBAX SB-C18 column (2.1×100mm, 1.8μm)

[0046] Mobile phase: A: acetonitrile B: 0.5% formic acid / water (V / V)

[0047] Flow rate: 0.4mL / min

[0048] Column temperature: 35°C

[0049] Injection volume: 1 μL

[0050] Detection wavelength: 283nm (hesperidin), 330nm

[0051] Gradient elution conditions: 0-2 min, 8% A; 2-10 min, 8%-18% A; 10-15 min, 18%-22% A; 15-20 min, 22%-37% A; 20-28 min, 37% A; 28-30 min, 37%-90% A

[0052] 1.4 Fingerprint Methodology Review

[0053] 1.4.1 Precision investigation

[0054] Take the sample of Zhangtouhong Chenpi with batch number A2312001-13, prepare the test solution according to the above 1.2.2 process, inject 6 times continuously, and record the fingerprint spectrum. Taking peak 7 (hesperidin) as the reference peak (S), the relative retention time RSD values ​​(relative standard deviation) of 21 common peaks (12 common peaks identified in 1.5, the same below) are all less than 1%, and the relative peak area RSD values ​​are all less than 5%, indicating that the instrument has good precision.

[0055] 1.4.2 Repeatability study

[0056] Take the sample of Zhangtouhong Chenpi with batch number A2312001-13, prepare 6 test solutions in parallel according to the above 1.2.2 process, inject the samples, and record the fingerprint spectrum. Taking peak 7 (hesperidin) as the reference peak (S), the relative retention time RSD values ​​of the 21 common peaks are all less than 1%, and the relative peak area RSD values ​​are all less than 5%, indicating that the method has good repeatability.

[0057] 1.4.3 Stability study

[0058] Take the sample of Zhangtouhong Chenpi with batch number A2312001-13, prepare the test solution according to the above 1.2.2 process, and inject it into the liquid chromatograph after leaving it at room temperature for 0h, 2h, 4h, 8h, 12h, and 24h, and record the fingerprint spectrum. Taking peak 7 (hesperidin) as the reference peak (S), the relative retention time RSD values ​​of 21 common peaks are calculated to be less than 1%, and the relative peak area RSD values ​​are less than 5%. The test solution has good stability within 24h.

[0059] 1.5 Establishment of fingerprint and identification of common peaks

[0060] According to the chromatographic analysis method established in 1.3, 1 μL of each of the 16 batches of Zhangtouhong Chenpi test solution (prepared according to the above 1.2.2 process) was accurately drawn and injected into the ultra-high performance liquid chromatograph to record the chromatographic peak information. An overlay graph was generated in the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", as shown in the figure. Figure 1 As shown ( Figure 1 From bottom to top, S1-S16 correspond to the red tangerine peels with batch numbers A2312001-1 to A2312001-16, and R corresponds to the reference fingerprint R). The reference fingerprint R is generated, such as Figure 2 As shown. There are 21 peaks in total. Liquid chromatography-mass spectrometry technology was used, combined with literature ([1] Zhang J, Wu X, Qiu J, et al. Comprehensive Comparison on the Chemical Profile of Guang Chen Pi at Different Ripeness Stages Using Untargeted and Pseudotargeted Metabolomics. J Agric Food Chem. 2020 Aug 5; 68(31): 8483-8495. [2] Zhang Ke, Xu Xia, Li Ting, et al. Analysis of the chemical composition group of dried tangerine peel using UHPLC-IT-TOF-MS [J]. Chinese Journal of Traditional Chinese Medicine, 2020, 45(04): 899-909. etc.) and databases (SciFinder n , ChemSpider, etc.) and the reference substance, 21 were identified, and the results are shown in Table 2. According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, 21 common peaks were matched for similarity, and the reference fingerprint of Zhangtouhongchenpi was used as a reference to calculate the similarity of 16 batches of samples with the reference fingerprint, all of which were greater than 0.95, and the results are shown in Table 3.

[0061] Table 2 Common peak identification results of Zhangtouhong Chenpi fingerprint

[0062]

[0063]

[0064] Table 3 Similarity between 16 batches of samples and reference spectra

[0065] serial number Similarity serial number Similarity A2312001-1 0.998 A2312001-9 0.998 A2312001-2 0.997 A2312001-10 0.985 A2312001-3 0.996 A2312001-11 0.996 A2312001-4 0.999 A2312001-12 0.995 A2312001-5 0.992 A2312001-13 0.996 A2312001-6 0.998 A2312001-14 0.998 A2312001-7 0.998 A2312001-15 0.997 A2312001-8 0.998 A2312001-16 0.998

[0066] Example 2: Establishment of UPLC content determination method for Zhangtouhong tangerine peel

[0067] 2.1 Instruments, reagents, test drugs and their sources

[0068] Same as "1.1 Instruments, reagents, test drugs and sources" in Example 1

[0069] 2.2 Solution preparation

[0070] 2.2.1 Preparation of reference solution

[0071] Same as "1.2.1 Preparation of solution" in Example 1.2

[0072] 2.2.2 Preparation of test solution

[0073] Same as "1.2.2 Preparation of test solution" in Example 1.2

[0074] 2.3 Chromatographic conditions

[0075] Same as "1.3 Chromatographic conditions" in Example 1.3

[0076] 2.4 Study on content determination methodology

[0077] 2.4.1 Precision investigation

[0078] Take the mixed reference solution under "2.1.1" and inject it continuously for 6 times according to the chromatographic conditions under "2.3". Record the peak areas of hesperidin, citrus aurantifolia, nobiletin, tangeretin and 5-demethylnobiletin. The results show that the RSD values ​​of the peak areas of hesperidin, citrus aurantifolia, nobiletin, tangeretin and 5-demethylnobiletin are all less than 1%, indicating that the instrument has good precision.

[0079] 2.4.2 Repeatability study

[0080] Take the camphor red tangerine peel sample with batch number A2312001-13, prepare 6 test solutions in parallel according to the conditions under "2.2.2", and analyze them according to the chromatographic conditions under "2.3". Record the peak areas of hesperidin, sweet orange flavonoids, nobiletin, tangeretin and 5-demethylnobiletin. The RSD values ​​of the peak areas of each component were calculated to be less than 2%, indicating that the method had good repeatability.

[0081] 2.4.3 Stability study

[0082] Take the sample solution of Camphor Red Tangerine Peel with batch number A2312001-13, and place it at room temperature for 0h, 2h, 4h, 8h, 12h, and 24h, respectively, and analyze the peak areas of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin to investigate the room temperature stability of the sample solution. The results showed that the RSD of the peak areas of the five index components was less than 1%, indicating that the sample solution had good stability within 24h, and the results are shown in Table 4.

[0083] Table 4 Results of the precision and stability test of content determination

[0084] name Precision (RSD%) Repeatability (RSD%) Stability (RSD%) Hesperidin 0.38 1.1 0.52 Sweet orange flavonoids 0.43 1.1 0.90 Nobiletin 0.35 1.3 0.37 Tangeretin 0.31 1.5 0.36 5-Demethylnobiletin 0.14 1.6 0.67

[0085] 2.4.4 Investigation of linear relationship

[0086] Take appropriate amount of each reference substance, weigh accurately, and prepare mixed reference substance stock solution containing 390.2460 mg of hesperidin, 7.8480 mg of sweet orange flavonoids, 81.6552 mg of nobiletin, 50.2462 mg of tangeretin, and 11.8200 mg of 5-demethylnobiletin per 1 mL. Accurately measure appropriate amount of mixed reference substance stock solution, dilute with methanol 1, 1.25, 1.67, 2.5, 5, 10, 25 times, respectively, to obtain mixed reference substance solutions of series concentrations, inject and analyze, perform linear regression on solution concentration X with peak area Y, and form standard curves of five quantitative indicators. The results are shown in Table 5. Figure 3 .

[0087] Table 5 Standard curves of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin

[0088] name Linear range (μg / mL) Regression equation <![CDATA[R 2 ]]> Hesperidin 15.61～390.2 <![CDATA[Y=4.732×10 3 X+4241.40]]> 0.998 Sweet orange flavonoids 0.3139～7.848 <![CDATA[Y=1.075×10 4 X+600.68]]> 0.999 Nobiletin 3.266～81.66 <![CDATA[Y=9.348×10 3 X+1850.89]]> 0.999 Tangeretin 2.010～50.25 <![CDATA[Y=1.075×10 4 X+1884.50]]> 0.999 5-Demethylnobiletin 0.4728～11.82 <![CDATA[Y=7.389×10 4 X+283.14]]> 0.999

[0089] 2.4.5 Sample recovery experiment

[0090] Accurately weigh about 0.1 g of a sample of Camphor Red Tangerine Peel with a known content of batch number A2312001-13, and accurately add known amounts of five reference substance mixed solutions at a concentration level of 100% of hesperidin (39005.39 μg per gram of sample), sweet orange flavonoids (436.43 μg per gram of sample), nobiletin (5593.37 μg per gram of sample), tangeretin (3916.02 μg per gram of sample), and 5-demethylnobiletin (760.16 μg per gram of sample). Prepare 6 test solution preparation methods in 1.2.2, and inject and analyze them separately. The recoveries of quantitative indicators (recovery % = (measured amount - content in sample) / amount of reference substance added * 100%) are all within 92-105%, and the RSD is less than 3%. The results are shown in Table 6.

[0091] Table 6 Recovery rates of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin

[0092]

[0093] 2.4.6 Intermediate precision test

[0094] In the same laboratory, different analysts took the batch number A2312001-13 of the red tangerine peel sample and prepared 6 test solutions for determination using different instruments. The RSD of the contents of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin were calculated (Table 7). The results showed that the RSD of the contents of the five flavonoid components was less than 2%, and the intermediate precision of the method was good.

[0095] Table 6 Intermediate precision of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin

[0096]

[0097] 2.5 Calculation of correction factors for hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin

[0098] Take the mixed reference solution under "2.1.1" and inject it 6 times in succession according to the chromatographic conditions under "2.3" to calculate the correction factor. The mean and RSD values ​​are shown in Table 7. The results show that the RSD values ​​of the correction factors of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin are all less than 2%, and the results meet the requirements.

[0099] Table 7 Correction factors for hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin

[0100]

[0101]

[0102] 2.6 Content determination results of each batch of samples

[0103] Take 16 batches of Zhangtouhong Chenpi samples, determine them according to the established chromatographic method, and record the chromatograms. Figure 4 As shown, from top to bottom are the chromatograms of the mixed reference solution (A) and the chromatograms of the Zhangtouhong Chenpi sample (B). Among them A i Indicates the peak area of ​​the component to be tested in the sample, Rf indicates the correction factor, m indicates the sample weight, and the correction factor = the peak area of ​​the reference substance A R / Concentration of reference substance C R), the contents of hesperidin, sweet orange flavonoids, nobiletin, tangeretin and 5-demethylnobiletin in the samples of Camphor Red Tangerine Peel were calculated. The contents of the five components determined are shown in Table 8. The results showed that the measured range of hesperidin was 3.25% to 4.80%, the SD value was 0.47, and the lower limit of the mean minus 3 times SD was 2.59%; the measured range of sweet orange flavonoids was 0.029% to 0.052%, the SD value was 0.006, and the lower limit of the mean minus 3 times SD was 0.025%; the measured range of nobiletin was 0.44% to 0.75%, the SD value was 0.07, and the lower limit of the mean minus 3 times SD was 0.035%; the measured range of tangerine peel was 0.25% to 0.45%, the SD value was 0.06, and the lower limit of the mean minus 3 times SD was 0.17%; the measured range of 5-demethylnobiletin was 0.060% to 0.116%, the SD value was 0.013, and the lower limit of the mean minus 3 times SD was 0.043%. Therefore, it is stipulated that the sample of Zhangtouhong Chenpi contains hesperidin (C 28 H 34 O 15 ) shall not be less than 2.59%; containing sweet orange flavonoids (C 20 H 20 O7) shall not be less than 0.025%; containing nobiletin (C 21 H 22 O8) shall not be less than 0.35%; containing tangeretin (C 20 H 20 O7) shall not be less than 0.17%; 5-demethylnobiletin (C 20 H 20 O8) shall not be less than 0.043%

[0104] Table 8 Content determination of each batch of samples of Zhangtouhong Chenpi (SD: standard deviation of samples)

[0105]

[0106]

[0107] Example 3: Application of UPLC quantitative fingerprint of red tangerine peel

[0108] 3.1 Instruments, reagents, test drugs and their sources

[0109] Instruments: Waters Acquity I Class ultra-high performance liquid chromatograph; Agilent Infinity II 1290-6545Q-TOF LC-MS Agilent ultra-high performance liquid chromatograph-quadrupole time-of-flight mass spectrometer; ML204T / 02 1 / 10,000 analytical balance; XSE105 1 / 100,000 analytical balance; KQ-500DV sonicator; Waters UPLC-DAD chromatographic conditions ZORBAXSB-C18 column (2.1×100 mm, 1.8 μm); SOrvall ST8 high-speed centrifuge;

[0110] Reagents: acetonitrile (Thermo) and formic acid (Thermo) were of mass spectrometry grade; formic acid (Macklin) and acetonitrile (Fisher) were of chromatography grade; methanol (Energy Chemical) was of analytical grade; ultrapure water;

[0111] Test drugs: Reference substances hesperidin (batch number 110721-202220, mass fraction 97.2%), nobiletin (batch number 112055-202102, mass fraction 99.7%), and tangeretin (batch number 112054-202102, mass fraction 99.7%) were purchased from China Food and Drug Inspection Institutes, 5-demethylnobiletin (batch number PS011601, mass fraction 98.5%) and sweet orange flavonoids (batch number PS011273, mass fraction 98.0%) were purchased from Pusi Biotechnology Co., Ltd.;

[0112] The test sample was identified as C. reticulate'Zhangshuensis'. Specific information is shown in Table 9.

[0113] Table 9 Information on samples of red tangerine peel

[0114] batch number Origin Name of medicinal material source Origin A2312001-17 Camphor red Tangerine peel Origin collection Xingan County, Ji'an City, Jiangxi Province A2312001-18 Camphor red Tangerine peel Origin collection Xingan County, Ji'an City, Jiangxi Province A2312001-19 Camphor red Tangerine peel Origin collection Xingan County, Ji'an City, Jiangxi Province

[0115] 3.2 Solution preparation

[0116] 3.2.1 Preparation of reference solution

[0117] Same as "1.2.1 Preparation of solution" in Example 1.2

[0118] 3.2.2 Preparation of test solution

[0119] Same as "1.2.2 Preparation of test solution" in Example 1.2

[0120] 3.3 Chromatographic conditions

[0121] Same as "1.3 Chromatographic conditions" in Example 1.3

[0122] 3.4 Application of the quantitative fingerprint of tangerine peel

[0123] 3.4.1 Application of the fingerprint method of Zhangtouhong Chenpi

[0124] According to the chromatographic conditions in 3.3, 1 μL of each of the three batches of Zhangtouhong Chenpi test solution (prepared according to the test solution in 3.2.2 above) was accurately drawn and injected into the ultra-high performance liquid chromatograph to record the chromatographic peak information. The retention time of the Zhangtouhong Chenpi sample and the reference peak of the reference substance corresponded to each other. According to the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine, the similarity of the test sample and the reference fingerprint was matched with 21 common peaks. The results are shown in Table 10. The similarity between the fingerprint of the test sample and the reference fingerprint was greater than 0.95, and the results met the requirements.

[0125] Table 10 Similarity results of the chromatograms of the test samples and the fingerprints of the control

[0126] batch number Similarity A2312001-17 0.997 A2312001-18 0.998 A2312001-19 0.998

[0127] 3.4.2 Application of the method for determining the content of Zhangtouhong tangerine peel

[0128] According to the chromatographic conditions in 3.3, accurately aspirate the reference solution and the test solution, perform liquid phase analysis, determine the corresponding peak areas, and use the external standard method ( Among them A i ( represents the peak area of ​​the component to be measured in the sample, Rf represents the correction factor, m represents the sample weight, and the dilution factor is 25) The contents of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in the samples of Camphor Red Tangerine Peel were calculated. The contents of the five components measured are shown in Table 11. The three batches of Camphor Red Tangerine Peel samples contained 4.23%, 4.19%, and 3.82% hesperidin, respectively; 0.040%, 0.046%, and 0.045% sweet orange flavonoids, respectively; 0.51%, 0.62%, and 0.63% nobiletin, respectively; 0.26%, 0.39%, and 0.37% tangeretin, respectively; and 0.073%, 0.092%, and 0.095% 5-demethylnobiletin, respectively, all meeting the requirements.

[0129] Table 11 Test sample content determination results

[0130]

[0131]

[0132] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A UPLC quantitative fingerprint detection method for red tangerine peel, characterized in that: The quantitative fingerprint detection method comprises the following steps: S11, preparation of mixed reference solution: taking hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin as reference substances, respectively adding methanol or ethanol with a volume concentration of 50% to 100% to prepare a mixed reference solution; S12, preparation of the test solution of Zhangtouhong tangerine peel: prepare a sample of Zhangtouhong tangerine peel into powder, add a methanol solvent or an ethanol solvent with a volume concentration of 50% to 100% according to a solid-liquid ratio of 1:25 to 1:500, treat with ultrasound or heating reflux for 10min to 60min, make up the lost weight with methanol solvent or ethanol solvent, shake well, centrifuge, and filter the supernatant to obtain the Zhangtouhong tangerine peel test solution; S13, UPLC analysis: prepare at least 3 groups of different batches of camphor red tangerine peel test solution according to S12 method, draw each group of camphor red tangerine peel test solution and mixed reference solution into ultra-high performance liquid chromatography, record the liquid chromatograms of the reference and test samples respectively, and record the peak areas of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in the reference and test samples; S14, establishment of fingerprint: import the liquid chromatograms of each group of camphor red tangerine peel test samples obtained in S13 into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", match the common peaks, obtain the control fingerprint, identify the common peaks, and establish the camphor red tangerine peel fingerprint; S15, calculating the content of the components of the red tangerine peel of Zhangtouhong: according to the peak area of ​​each component recorded in S13, the content of hesperidin, sweet orange flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in the red tangerine peel of Zhangtouhong was calculated by external standard method; wherein, In the formula, A i represents the peak area of ​​the component to be tested in the sample, Rf represents the correction factor, and m represents the sample weight; A R Indicates the peak area of ​​the component in the reference substance, C R Indicates the concentration of the ingredient in the reference substance.

2. A UPLC quantitative fingerprint detection method for tangerine peel of Zhangtouhong according to claim 1, characterized in that, The concentrations of the components in the mixed reference solution of step S11 are: hesperidin 200 μg / mL to 300 μg / mL, sweet orange flavonoids 1 μg / mL to 10 μg / mL, nobiletin 20 μg / mL to 60 μg / mL, tangeretin 10 μg / mL to 40 μg / mL, and 5-demethylnobiletin 1 μg / mL to 10 μg / mL.

3. A UPLC quantitative fingerprint detection method for tangerine peel of Zhangtouhong according to claim 1, characterized in that, The solid-liquid ratio of step S12 is 1:100 to 1:

150.

4. A UPLC quantitative fingerprint detection method for tangerine peel of Zhangtouhong according to claim 1, characterized in that, The conditions for UPLC analysis in step S13 include: The chromatographic column is ZORBAX SB-C18, the column size is 2.1×100 mm, the column filler particle size is 1.8 μm, the column temperature is 30-40° C., the flow rate is 0.3-0.5 mL / min, the injection volume is 0.5-2 μL, the detection wavelengths are 283 nm and 330 nm, acetonitrile is used as mobile phase A, and formic acid with a volume percentage concentration of 0.1-1.0% is used as mobile phase B for gradient elution; the elution gradient conditions are: 0-2 min, 8% A; 2-10 min, 8%-18% A; 10-15 min, 18%-22% A; 15-20 min, 22%-37% A; 20-28 min, 37% A; 28-30 min, 37%-90% A.

5. A UPLC quantitative fingerprint detection method for tangerine peel of Zhangtouhong according to claim 1, characterized in that, In the step S13, the number of the test solutions of Camphor-head red tangerine peel is no less than 10 groups.

6. A UPLC quantitative fingerprint detection method for tangerine peel of Zhangtouhong according to claim 1, characterized in that, There are 21 common peaks in the control fingerprint of step S14, of which peak 1 is 3-O-caffeoylquinic acid, peak 2 is vecinin-2, peak 3 is stellarin-2, peak 4 is limonin 17-β-D-glucopyranoside, peak 5 is naringin, peak 6 is hesperidin, peak 7 is dicaffeoylquinic acid, peak 8 is nomilin Peak 9 is methyl dicaffeoylquinate, Peak 10 is fennel glycoside, Peak 11 is natsudaidain-3-O-(5-α-glucosyl-HMG)-β-glucoside, Peak 12 is hydroxy-trimethoxyflavone, Peak 13 is hydroxy-tetramethoxyflavone, Peak 14 is sweet orange flavone, Peak 15 is 6-demethoxytangeretin, Peak 16 is nobiletin, Peak 17 is 4',5,6,7-tetramethoxyflavone, Peak 18 is 3,3',4',5,6,7,8-heptamethoxyflavone, Peak 19 is 5-hydroxy-6,7,3',4'-tetramethoxyflavone, Peak 20 is tangeretin, Peak 21 is 5-demethylnobiletin.

7. A UPLC quantitative fingerprint detection method for tangerine peel of Zhangtouhong according to claim 1 or 6, characterized in that, In the step S14, the chromatogram of the camphor red tangerine peel test solution at a wavelength of 330 nm should show 21 characteristic peaks, of which 5 peaks correspond to the retention time of the reference peaks of the reference substance, and the similarity is calculated based on the 21 common peaks, and the similarity between the test sample chromatogram and the reference chromatogram is not less than 0.

95.

8. A UPLC quantitative fingerprint detection method for tangerine peel of Zhangtouhong according to claim 1, characterized in that, In the step S15, the contents of citrus flavonoids, nobiletin, tangeretin, and 5-demethylnobiletin in the camphor-red tangerine peel are calculated by the external standard method based on the peak area in the chromatogram at a detection wavelength of 330 nm, and the content of hesperidin is calculated by the external standard method based on the peak area in the chromatogram at a detection wavelength of 283 nm.

9. A UPLC quantitative fingerprint detection method for red tangerine peel of Zhangtouhong according to claim 1 or 8, characterized in that, In step S15, the camphor red tangerine peel sample contains hesperidin (C 28 H 34 O 15 ) shall not be less than 2.59%; containing sweet orange flavonoids (C 20 H 20 O7) shall not be less than 0.025%; containing nobiletin (C 21 H 22 O8) shall not be less than 0.35%; containing tangeretin (C 20 H 20 O7) shall not be less than 0.17%; 5-demethylnobiletin (C 20 H 20 O8) shall not be less than 0.043%.

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