A detection method for UPLC quantitative fingerprint of zhangtou red pericarpium citri reticulatae

Abstract

The application provides a kind of zhangtou red pericarpium citri reticulatae UPLC quantitative fingerprint detection method, belongs to the quality detection technical field of traditional Chinese medicine.The detection method is prepared by reference solution, preparation of test solution, using ultra high performance liquid chromatography analysis, multiple batches of liquid chromatogram is introduced into "traditional Chinese medicine chromatographic fingerprint similarity evaluation system", 21 common peaks are identified, similarity matching is carried out with 21 common peaks, and the control spectrum of zhangtou red pericarpium citri reticulatae is generated.On this basis, combined with hesperidin, sweet orange flavone, chuan chenpisi, tangeretin, 5-dimethyl chuan chenpisi 5 flavonoid content determination, zhangtou red pericarpium citri reticulatae quantitative fingerprint is established.The application realizes that fingerprint detection and the content determination of 5 flavonoids of zhangtou red pericarpium citri reticulatae can be completed at the same time through one analysis, can quickly, comprehensively and accurately evaluate the quality of zhangtou red pericarpium citri reticulatae, has good reproducibility and stability, and can provide technical support for the uniformity and stability of zhangtou red pericarpium citri reticulatae quality.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology of traditional Chinese medicine, and specifically relates to a UPLC quantitative fingerprint detection method for camphor-headed red tangerine peel. Background Technology

[0002] Camphor Red C. reticulate ' Zhangshuensis As a cultivated variety of dried tangerine peel, it is a superior variety resulting from a natural hybridization of Jiangxi Zhou Shang red tangerine and local sour orange, belonging to a unique local dried tangerine peel variety of Zhangshu City, Jiangxi Province. Zhangtou Red Tangerine Peel was included in the "Ten Delicacies of Jiangxi" list in 2020. It is a traditional Chinese medicine that is both food and medicine, and can be used in medicine and as a tea. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and has a huge market prospect.

[0003] The chemical composition of traditional Chinese medicine (TCM) is extremely complex, with diverse chemical structures and a wide range of polarity distributions. Traditional microscopic identification and physicochemical analysis are insufficient to characterize the quality of TCM. Furthermore, routine 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 in the literature regarding UPLC fingerprinting of Zhangtouhong Chenpi (Citrus aurantium var. camphora) and the determination of the content of multiple flavonoids. Currently, no literature has publicly applied quantitative fingerprinting methods to the quality control of Zhangtouhong Chenpi, and a relatively systematic quality control method has not yet been established, which seriously restricts the development of the Zhangtouhong Chenpi industry. Summary of the Invention

[0004] Therefore, the present invention aims to provide a UPLC quantitative fingerprinting method for detecting camphor-headed red tangerine peel, which addresses the problems of high operating costs and difficulties in promotion in existing technologies where developing fingerprint spectra and quantifying multiple indicators typically requires the development of multiple analytical methods. The quantitative fingerprinting method utilizes a single analytical method to simultaneously determine the fingerprint spectrum and the content of multiple indicators, significantly improving detection efficiency and reducing detection costs.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for detecting quantitative fingerprint spectrum of camphor red tangerine peel by UPLC, specifically including steps S11 to S15.

[0007] S11: Preparation of reference solutions: Take hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin as reference standards, and add 50-100% methanol (V / V) or 50-100% ethanol (V / V) to prepare mixed reference solutions.

[0008] S12: Preparation of the test solution: Take an appropriate amount of camphor red tangerine peel sample, accurately weigh it, and put it into a stoppered conical flask. Add methanol solvent with a volume concentration of 50%~100% or ethanol solvent with a volume concentration of 50%~100%. The material-liquid ratio (the ratio of sample to methanol solvent or ethanol solvent) is 1:25~1:500 g / mL (preferably 1:100~1:150 g / mL). Sonicate or reflux for 10~60 min, make up for the weight loss with solvent, shake well, centrifuge, and filter the supernatant to obtain the camphor red tangerine peel test solution.

[0009] S13: Ultra-high performance liquid chromatography (UPLC) analysis: Prepare three or more (preferably more than ten, more preferably more than twenty) different batches of camphor red tangerine peel test solutions according to the method in step 2. Take the reference solution and test solution respectively and inject them into the ultra-high performance liquid chromatograph. Record the liquid chromatograms of the reference solution and the test solution respectively, and record the peak areas of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin and 5-demethylnorihesperidin in the reference solution and the test solution.

[0010] S14: Establishment of fingerprint spectrum: Import the liquid chromatograms of at least 3 (preferably more than 10) different batches of camphor red tangerine peel test samples into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition", match common peaks, obtain reference fingerprint spectrum, identify common peaks, and establish the fingerprint spectrum of camphor red tangerine peel.

[0011] S15: Establishment of content determination method: Based on the peak areas of each component recorded in S13, the content determination method is determined by external standard method ( A i Rf represents the peak area of ​​the analyte in the sample, Rf represents the correction factor, and m represents the sample weight. Calculate the contents of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin in the sample of camphor red tangerine peel;

[0012] Furthermore, the concentrations of the mixed reference solution in step S11 are as follows: 200-300 μg of hesperidin, 1-10 μg of sweet orange flavonoids, 20-60 μg of norihesperidin, 10-40 μg of tangeretin, and 1-10 μg of 5-demethylnorihesperidin per 1 mL.

[0013] Further, the ultra-high performance liquid chromatography (UHPLC) conditions described in step S13 are as follows: the chromatographic column is a ZORBAX SB-C18 (2.1×100 mm, 1.8 µm), the column temperature is 30~40 ℃, the flow rate is 0.3~0.5 mL / min, the injection volume is 0.5~2 μL, the detection wavelength is 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.

[0014] Furthermore, the comparative fingerprint spectrum in step S14 contains 21 common peaks, including peak 1 (3-O-caffeoylquinic acid), peak 2 (vezin-2), peak 3 (stellarin-2), peak 4 (Limonin 17-β-D-glucopyranoside), peak 5 (naringin), peak 6 (hesperidin), peak 7 (dicaffeoylquinic acid), and peak 8 (nomilin). Peak 17-β-D-glucopyranoside, peak 9 is methyl dicaffeoylquinic acid, peak 10 is sennaoside, 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 flavonoid, peak 15 is 6-demethoxyhesperidin, peak 16 is norihesperidin, 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 hesperidin, and peak 21 is 5-demethylnorihesperidin.

[0015] Furthermore, in 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 should correspond to the retention time of the reference peak of the reference standard. The similarity is calculated based on the 21 common peaks, and the similarity between the chromatogram of the test sample and the reference chromatogram is not less than 0.95.

[0016] Furthermore, in step S15, the contents of sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin in the test solution of 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 contents of hesperidin are calculated by the external standard method based on the peak area in the chromatogram at a detection wavelength of 283 nm.

[0017] Furthermore, in step S15, the hesperidin (C...) in the sample solution of camphor red tangerine peel... 28 H 34 O 15 The content shall not be less than 2.59%; it contains sweet orange flavonoids (C 20 H 20 O7) must not be less than 0.025%; containing norihesperidin (C 21 H 22 O8) must not be less than 0.35%; containing hesperidin (C 20 H 20 O7) shall not be less than 0.17%; containing 5-demethylhesperidin (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 using the 21 common peaks to generate a reference spectrum of camphor red tangerine peel. Combined with the determination of the content of five flavonoid components, namely hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin, a quantitative fingerprint spectrum of camphor red tangerine peel was established.

[0019] This invention enables simultaneous fingerprint spectrum detection and content determination of camphor red tangerine peel in a single analysis, allowing for rapid, comprehensive, and accurate evaluation of its quality and providing technical support for ensuring the uniformity of its quality.

[0020] The present invention has the following advantages:

[0021] 1. The UPLC quantitative fingerprint spectrum detection method for camphor red tangerine peel provided by the present invention can realize the fingerprint spectrum detection and the determination of the content of five flavonoid components in a single analysis, which can rapidly, comprehensively and accurately evaluate the quality of camphor red tangerine peel and provide technical support for the quality uniformity of camphor red tangerine peel;

[0022] 2. The UPLC quantitative fingerprint detection method for camphor red tangerine peel provided by this invention uses the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine provided by the National Pharmacopoeia Commission to construct a control fingerprint spectrum for the measured fingerprint spectrum and calculate the similarity. The operation is convenient and fast, and the conclusions are objective and accurate.

[0023] 3. The UPLC quantitative fingerprinting detection method for camphor red tangerine peel provided by the present invention can effectively control the quality of camphor red tangerine peel by measuring five flavonoid components: hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin.

[0024] 4. The UPLC quantitative fingerprinting method for detecting camphor red tangerine peel provided by this invention has conducted methodological investigations on the precision, repeatability, and stability of the fingerprinting, as well as the linearity, recovery rate, precision, repeatability, and stability of the content determination. The method is accurate and reliable, providing a more comprehensive quality control method for camphor red tangerine peel. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0026] Figure 1 This is a superimposed image of the fingerprint spectra of 16 batches of camphor red tangerine peel from Example 1;

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

[0028] Figure 3 The standard curves for hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin in Example 2 are shown below.

[0029] Figure 4 This is a comparison chart of the content determination of the mixed reference standard and camphor red tangerine peel in Example 2. Detailed Implementation

[0030] The following detailed description of the UPLC quantitative fingerprint detection method for camphor red tangerine peel of the present invention is provided in conjunction with specific embodiments.

[0031] Example 1: Establishment of UPLC fingerprinting method for camphor-headed red tangerine peel

[0032] 1.1 Instruments, reagents, and sources

[0033] Instruments: Waters Acquity I Class UHPLC; Agilent Infinity II 1290-6545 Q-TOF LC-MS; ML204T / 02 0.01% analytical balance; XSE105 0.01% analytical balance; KQ-500DV ultrasonic instrument; Waters UPLC-DAD chromatographic conditions; ZORBAX SB-C18 column (2.1×100 mm, 1.8 μm); SOrvall ST8 high-speed centrifuge;

[0034] Reagents: Acetonitrile (Thermo) and formic acid (Thermo) are mass spectrometry grade; formic acid (Macklin) and acetonitrile (Fisher) are chromatographic grade; methanol (Energy Chemical) is analytical grade; ultrapure water;

[0035] Test reagents: The reference standards hesperidin (batch number 110721-202220, mass fraction 97.2%), norihesperidin (batch number 112055-202102, mass fraction 99.7%), and citrus peelin (batch number 112054-202102, mass fraction 99.7%) were all purchased from the National Institutes for Food and Drug Control. 5-Demethylnorihesperidin (batch number PS011601, mass fraction 98.5%) and sweet orange flavonoids (batch number PS011273, mass fraction 98.0%) were all purchased from Pusi Biotechnology Co., Ltd.

[0036] The test sample was identified as C. reticulate. Zhangshuensis For details, please refer to Table 1.

[0037] Table 1. Sample Information of Camphor Red Tangerine Peel

[0038]

[0039] 1.2 Solution Preparation

[0040] 1.2.1 Preparation of reference solution

[0041] Preparation of mixed standard solution: Take appropriate amounts of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 234.1 μg of hesperidin, 4.9 μg of sweet orange flavonoids, 40.8 μg of norihesperidin, 20.9 μg of tangeretin, and 4.9 μg of 5-demethylnorihesperidin per mL.

[0042] 1.2.2 Preparation of test solution

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

[0044] 1.3 Chromatographic conditions

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

[0046] Mobile phase: A: Acetonitrile; B: 0.5% formic acid / water (v / v)

[0047] Flow rate: 0.4 mL / min

[0048] Column temperature: 35 °C

[0049] Injection volume: 1 µL

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

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

[0052] 1.4 Investigation of Fingerprint Mapping Methodology

[0053] 1.4.1 Precision Examination

[0054] A sample of camphor red tangerine peel with batch number A2312001-13 was taken, and the test solution was prepared according to the procedure in 1.2.2 above. The sample was injected 6 times consecutively, and the fingerprint spectrum was recorded. Using peak 7 (hesperidin) as the reference peak (S), the relative retention time RSD (relative standard deviation) values ​​of the 21 common peaks (12 common peaks identified in 1.5, the same below) were all less than 1%, and the relative peak area RSD values ​​were all less than 5%, indicating that the instrument precision was good.

[0055] 1.4.2 Repeatability Test

[0056] A sample of camphor red tangerine peel with batch number A2312001-13 was used. Six test solutions were prepared in parallel according to the procedure described in 1.2.2 above. The samples were injected, and the fingerprint chromatograms were recorded. Using peak 7 (hesperidin) as the reference peak (S), the relative retention time RSD values ​​of the 21 common peaks were all less than 1%, and the relative peak area RSD values ​​were all less than 5%, indicating that the method has good repeatability.

[0057] 1.4.3 Stability Test

[0058] A sample of camphor-root red tangerine peel with batch number A2312001-13 was taken, and a test solution was prepared according to the procedure in 1.2.2 above. After being placed at room temperature for 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, the solution was injected into the liquid chromatograph, and the fingerprint chromatogram was recorded. Using peak 7 (hesperidin) as the reference peak (S), the relative retention time RSD values ​​of the 21 common peaks were all less than 1%, and the relative peak area RSD values ​​were all less than 5%, indicating that the test solution had good stability within 24 h.

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

[0060] Following the established chromatographic analysis method in 1.3, precisely pipette 1 μL of each of 16 batches of *Citrus reticulata* var. *camphor* (prepared according to the procedure in 1.2.2 above) into the ultra-high performance liquid chromatograph (UHPLC), and record the chromatographic peak information. Generate an overlay image in the *Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012*, as shown below. Figure 1 As shown ( Figure 1 From bottom to top, S1-S16 correspond to batch numbers A2312001-1 to A2312001-16 of camphor-headed red tangerine peel, and R corresponds to the control fingerprint spectrum R). A control fingerprint spectrum R is generated, as shown below. Figure 2 As shown. There are 21 peaks in total. Liquid chromatography-mass spectrometry (LC-MS) 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 chemical components of tangerine peel by UHPLC-IT-TOF-MS[J]. Chinese Journal of Traditional Chinese Medicine, 2020, 45 (04): 899-909. etc.), database (SciFinder etc.). n The samples were compared with reference standards (such as ChemSpider, etc.) and identified 21 peaks. The results are shown in Table 2. According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity was matched with the 21 common peaks. The fingerprint of the 16 batches of samples and the reference fingerprint were used as a reference. The similarity was calculated between the fingerprint of the chromatographic fingerprint of the 16 batches of samples and the reference fingerprint. All of them were greater than 0.95. The results are shown in Table 3.

[0061] Table 2. Results of Common Peak Identification in the Fingerprint Spectra of Camphor Red Tangerine Peel

[0062]

[0063] Table 3. Similarity between 16 batches of samples and the control chromatograms

[0064]

[0065] Example 2: Establishment of UPLC method for determining the content of camphor-root red tangerine peel

[0066] 2.1 Instruments, reagents, and sources

[0067] Same as in Example 1, "1.1 Instruments, Reagents, Tests and Sources"

[0068] 2.2 Solution Preparation

[0069] 2.2.1 Preparation of reference solution

[0070] Same as in Example 1.2, "1.2.1 Preparation of the Solution"

[0071] 2.2.2 Preparation of test solution

[0072] Same as "1.2.2 Preparation of Test Solution" in Example 1.2.

[0073] 2.3 Chromatographic conditions

[0074] Same as "1.3 Chromatographic Conditions" in Example 1.3

[0075] 2.4 Methodological Investigation of Content Determination

[0076] 2.4.1 Precision Examination

[0077] Take the mixed reference solution under section "2.1.1" and inject it 6 times consecutively under the chromatographic conditions under section "2.3". Record the peak areas of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin. The results show that the RSD values ​​of the peak areas of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin are all less than 1%, indicating that the instrument precision is good.

[0078] 2.4.2 Repeatability Test

[0079] Take a sample of camphor red tangerine peel with batch number A2312001-13, prepare 6 test solutions in parallel according to section "2.2.2", and analyze them according to the chromatographic conditions in section "2.3". Record the peak areas of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin. The RSD values ​​of the peak areas of each component are all less than 2%, indicating that the method has good repeatability.

[0080] 2.4.3 Stability Assessment

[0081] A sample solution of *Citrus reticulata* var. *camphora* (batch number A2312001-13) was taken and analyzed after being left at room temperature for 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The peak areas of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin were recorded to examine the room temperature stability of the sample solution. The results showed that the RSD of the peak areas of the five components was less than 1%, indicating that the sample solution had good stability within 24 h. The results are shown in Table 4.

[0082] Table 4 Results of the content determination and steady-state test

[0083]

[0084] 2.4.4 Examination of Linear Relationships

[0085] Accurately weigh appropriate amounts of each reference standard to prepare a mixed reference stock solution containing 390.2460 mg of hesperidin, 7.8480 mg of sweet orange flavonoids, 81.6552 mg of norepinephrine, 50.2462 mg of citrus terpenoid, and 11.8200 mg of 5-demethylnorepinephrine per 1 mL. Accurately measure appropriate amounts of the mixed reference stock solution and dilute it with methanol by 1, 1.25, 1.67, 2.5, 5, 10, and 25 times, respectively, to obtain a series of mixed reference solutions of different concentrations. Inject these solutions for analysis. Linear regression was performed on the peak area Y against the solution concentration X to generate standard curves for the five quantitative indicators. The results are shown in Table 5. Figure 3 .

[0086] Table 5 Standard curves for hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin

[0087]

[0088] 2.4.5 Recovery Experiment

[0089] Accurately weigh approximately 0.1 g of a sample of camphor red tangerine peel (batch number A2312001-13) with known content. Add known amounts of a mixed solution of five reference standards to the sample, ensuring 100% concentrations of hesperidin (39005.39 μg per g sample), sweet orange flavonoids (436.43 μg per g sample), norihesperidin (5593.37 μg per g sample), citrus terpenoids (3916.02 μg per g sample), and 5-demethylnorihesperidin (760.16 μg per g sample). Prepare six test solutions according to the test solution preparation method in section 1.2.2. Inject each solution into the solution for analysis. The recoveries of the quantitative indicators (recovery rate % = (analyzed amount - content in sample) / amount of added reference standard * 100%) were all within 92-105%, with RSD less than 3%. The results are shown in Table 6.

[0090] Table 6. Recovery rates of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin.

[0091]

[0092] 2.4.6 Intermediate Precision Test

[0093] In the same laboratory, at different times, different analysts collected batch number A2312001-13 of camphor red tangerine peel and prepared six test solutions. These solutions were then analyzed using different instruments to calculate the relative standard deviations (RSDs) of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin (Table 7). The results showed that the RSDs of all five flavonoid components were less than 2%, indicating good intermediate precision of the method.

[0094] Table 7 Intermediate Precision of Hesperidin, Sweet Orange Flavonoids, Norihesperidin, Citrulline, and 5-Demethylnorihesperidin

[0095]

[0096] 2.5 Calculation of correction factors for hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin

[0097] Take the mixed reference solution from section "2.1.1", and inject it six times consecutively under the chromatographic conditions from section "2.3". Calculate the correction factor ( The mean and RSD values ​​of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin are shown in Table 8. The results show that the correction factor RSD values ​​of hesperidin, sweet orange flavonoids, norihesperidin, and tangeretin are all less than 2%, which meets the requirements.

[0098] Table 8 Correction factors for hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin

[0099]

[0100] 2.6 Content determination results of each batch of samples

[0101] Sixteen batches of camphor-root red tangerine peel samples were collected and analyzed according to the established chromatographic method. The chromatograms were recorded as follows: Figure 4 As shown, from top to bottom, the chromatograms are of the mixed reference solution (A) and the sample of camphor red tangerine peel (B), obtained using the external standard method (…). A i Rf represents the peak area of ​​the analyte in the sample, m represents the correction factor, and the correction factor is equal to the peak area of ​​the reference standard (A). R / Reference concentration C RThe contents of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin in the sample of camphor red tangerine peel were calculated. The contents of the five components were shown in Table 9. The results showed that hesperidin's measured range was 3.25%–4.80%, with an SD value of 0.47 and a lower limit of 2.59% (mean minus 3 times SD); sweet orange flavonoids' measured range was 0.029%–0.052%, with an SD value of 0.006 and a lower limit of 0.025% (mean minus 3 times SD); norihesperidin's measured range was 0.44%–0.75%, with an SD value of 0.07 and a lower limit of 0.035% (mean minus 3 times SD); citrus terpene's measured range was 0.25%–0.45%, with an SD value of 0.06 and a lower limit of 0.17% (mean minus 3 times SD); and 5-demethylnorihesperidin's measured range was 0.060%–0.116%, with an SD value of 0.013 and a lower limit of 0.043% (mean minus 3 times SD). Therefore, it was stipulated that the *C. camphor* red tangerine peel sample contains hesperidin (C). 28 H 34 O 15 The content shall not be less than 2.59%; it contains sweet orange flavonoids (C 20 H 20 O7) must not be less than 0.025%; containing norihesperidin (C 21 H 22 O8) must not be less than 0.35%; containing hesperidin (C 20 H 20 O7) shall not be less than 0.17%; containing 5-demethylhesperidin (C 20 H 20 O8) shall not be less than 0.043%.

[0102] Table 9. Content determination of different batches of camphor red tangerine peel (SD: standard deviation of the sample)

[0103]

[0104] Example 3: Application of UPLC quantitative fingerprinting of camphor-headed red tangerine peel

[0105] 3.1 Instruments, reagents, and sources

[0106] Instruments: Waters Acquity I Class UHPLC; Agilent Infinity II 1290-6545 Q-TOF LC-MS; ML204T / 02 0.01% analytical balance; XSE105 0.01% analytical balance; KQ-500DV ultrasonic instrument; Waters UPLC-DAD chromatographic conditions; ZORBAX SB-C18 column (2.1×100 mm, 1.8 μm); SOrvall ST8 high-speed centrifuge;

[0107] Reagents: Acetonitrile (Thermo) and formic acid (Thermo) are mass spectrometry grade; formic acid (Macklin) and acetonitrile (Fisher) are chromatographic grade; methanol (Energy Chemical) is analytical grade; ultrapure water;

[0108] Test reagents: The reference standards hesperidin (batch number 110721-202220, mass fraction 97.2%), norihesperidin (batch number 112055-202102, mass fraction 99.7%), and citrus peelin (batch number 112054-202102, mass fraction 99.7%) were all purchased from the National Institutes for Food and Drug Control. 5-Demethylnorihesperidin (batch number PS011601, mass fraction 98.5%) and sweet orange flavonoids (batch number PS011273, mass fraction 98.0%) were all purchased from Pusi Biotechnology Co., Ltd.

[0109] The test sample was identified as C. reticulate. Zhangshuensis For details, please refer to Table 10.

[0110] Table 10 Information on Samples of Camphor Red Tangerine Peel

[0111]

[0112] 3.2 Solution Preparation

[0113] 3.2.1 Preparation of reference solution

[0114] Same as in Example 1.2, "1.2.1 Preparation of the Solution"

[0115] 3.2.2 Preparation of test solution

[0116] Same as "1.2.2 Preparation of Test Solution" in Example 1.2.

[0117] 3.3 Chromatographic conditions

[0118] Same as "1.3 Chromatographic Conditions" in Example 1.3

[0119] 3.4 Application of quantitative fingerprinting of camphor-headed red tangerine peel

[0120] 3.4.1 Application of the fingerprint spectroscopy method for camphor-headed red tangerine peel

[0121] According to the chromatographic conditions in 3.3, 1 μL of each of three batches of *Citrus reticulata* var. *camphorata* (prepared according to the test solution in 3.2.2 above) was precisely pipetted into the ultra-high performance liquid chromatograph, and the chromatographic peak information was recorded. The retention times of the tested *Citrus reticulata* var. *camphorata* samples corresponded to those of the reference standard. Using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the test sample and the reference fingerprint chromatogram was matched based on 21 common peaks. The results are shown in Table 11. The similarity between the fingerprint chromatograms of the test sample and the reference fingerprint chromatogram was greater than 0.95, and the results met the requirements.

[0122] Table 11 Similarity results between the chromatograms of the test sample and the reference fingerprint chromatograms

[0123]

[0124] 3.4.2 Application of the method for determining the content of camphor red tangerine peel

[0125] According to the chromatographic conditions in 3.3, accurately pipette the reference solution and the test solution separately, perform liquid chromatography analysis, determine the corresponding peak areas, and use the external standard method (…). A i The peak area of ​​the analyte in the sample is represented by Rf, the correction factor is represented by m, and the dilution factor is 25. The contents of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin in the samples of Zhangtouhong tangerine peel were calculated. The contents of the five components were shown in Table 12. The hesperidin contents of the three batches of Zhangtouhong tangerine peel samples were 4.23%, 4.19%, and 3.82%, respectively; the sweet orange flavonoid contents were 0.040%, 0.046%, and 0.045%, respectively; the norihesperidin contents were 0.51%, 0.62%, and 0.63%, respectively; the tangeretin contents were 0.26%, 0.39%, and 0.37%, respectively; and the 5-demethylnorihesperidin contents were 0.073%, 0.092%, and 0.095%, respectively. All of these contents met the requirements.

[0126] Table 12 Results of content determination of test sample

[0127]

[0128] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for quantitative fingerprint detection of camphor-headed red tangerine peel using UPLC, characterized in that, The quantitative fingerprint detection method includes the following steps: S11. Preparation of mixed reference solution: Take hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin as reference standards, and add methanol or ethanol with a volume concentration of 50%~100% to prepare a mixed reference solution. S12. Preparation of the test solution of camphor red tangerine peel: Take the camphor red tangerine peel sample and make it into powder. Add methanol or ethanol solvent with a volume concentration of 50% to 100% according to the solid-liquid ratio of 1:25 to 1:

500. Sonicate or heat and reflux for 10 min to 60 min. Make up the weight loss with methanol or ethanol solvent, shake well, centrifuge, take the supernatant and filter it to obtain the test solution of camphor red tangerine peel. S13, UPLC analysis: Prepare at least three different batches of camphor red tangerine peel test solutions according to method S12. Inject each group of camphor red tangerine peel test solutions and mixed reference solutions into an ultra-high performance liquid chromatograph. Record the liquid chromatograms of the reference and test samples respectively, and record the peak areas of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin in the reference and test samples. S14. Establishment of fingerprint spectrum: Import the liquid chromatograms of each group of camphor red tangerine peel test samples recorded in S13 into the "Similarity Evaluation System of Chromatographic Fingerprint Spectrum of Traditional Chinese Medicine", match common peaks to obtain the reference fingerprint spectrum, identify the common peaks, and establish the fingerprint spectrum of camphor red tangerine peel. S15. Calculate the content of components in camphor red tangerine peel: Based on the peak areas of each component recorded in S13, calculate the content of hesperidin, sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin in the camphor red tangerine peel sample using the external standard method; among which, the content of the components to be tested is... In the formula, A i Rf represents the peak area of ​​the analyte in the sample, Rf represents the correction factor, and m represents the sample weight. AR represents the peak area of ​​the component in the reference standard, and CR represents the concentration of the component in the reference standard. The conditions for UPLC analysis in step S13 include: The chromatographic column was a ZORBAX SB-C18 with a column size of 2.1 × 100 mm and a packing particle size of 1.8 µm. The column temperature was 30–40 °C, the flow rate was 0.3–0.5 mL / min, the injection volume was 0.5–2 μL, and the detection wavelengths were 283 nm and 330 nm. Acetonitrile was used as mobile phase A, and formic acid with a volume percentage concentration of 0.5% was used as mobile phase B for gradient elution. The gradient elution conditions were as follows: 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.

2. The method for detecting quantitative fingerprint spectroscopy of camphor-head red tangerine peel by UPLC according to claim 1, characterized in that, The concentrations of each component in the mixed reference solution in step S11 are as follows: hesperidin 200 μg / mL~300 μg / mL, sweet orange flavonoid 1 μg / mL~10 μg / mL, nobiletin 20 μg / mL~60 μg / mL, citrus 10 μg / mL~40 μg / mL, and 5-demethylnobiletin 1 μg / mL~10 μg / mL.

3. The method for detecting quantitative fingerprint spectrum of camphor-head red tangerine peel by UPLC according to claim 1, characterized in that, The solid-liquid ratio in step S12 is 1:100 to 1:

150.

4. The method for detecting quantitative fingerprint spectrum of camphor-headed red tangerine peel by UPLC according to claim 1, characterized in that, In step S13, there are no fewer than 10 sets of camphor red tangerine peel test solutions.

5. The method for detecting quantitative fingerprint spectrum of camphor-headed red tangerine peel by UPLC according to claim 1, characterized in that, The fingerprint spectrum obtained in step S14 contains 21 common peaks, including peak 1 (3-O-caffeoylquinic acid), peak 2 (vezin-2), peak 3 (stellarin-2), peak 4 (Limonin 17-β-D-glucopyranoside), peak 5 (naringin), peak 6 (hesperidin), peak 7 (dicaffeoylquinic acid), and peak 8 (nomilin). Peak 17-β-D-glucopyranoside, peak 9 is methyl dicaffeoylquinic acid, peak 10 is sennaoside, 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 flavonoid, peak 15 is 6-demethoxyhesperidin, peak 16 is norihesperidin, 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 hesperidin, and peak 21 is 5-demethylnorihesperidin.

6. The method for detecting quantitative fingerprint spectrum of camphor-head red tangerine peel by UPLC according to claim 1 or 5, characterized in that, In 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 peak. The similarity is calculated based on the 21 common peaks, and the similarity between the chromatogram of the test sample and the reference chromatogram is not less than 0.

95.

7. The method for detecting quantitative fingerprint spectrum of camphor-head red tangerine peel by UPLC according to claim 1, characterized in that, In step S15, the contents of sweet orange flavonoids, norihesperidin, tangeretin, and 5-demethylnorihesperidin 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 contents of hesperidin are calculated by the external standard method based on the peak area in the chromatogram at a detection wavelength of 283 nm.

8. The method for detecting quantitative fingerprint spectrum of camphor-head red tangerine peel by UPLC according to claim 1 or 7, characterized in that, In step S15, the camphor red tangerine peel sample shall contain not less than 2.59% hesperidin; not less than 0.025% sweet orange flavonoids; not less than 0.35% hesperidin; not less than 0.17% citrus peel; and not less than 0.043% 5-demethylhesperidin.

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