A detection method for orange and bitter orange formula granules

Abstract

The present invention relates to the technical field of detection of Chinese medicine formula granules, and discloses a detection method for orange and citrus aurantium formula granules. The method comprises first comparing the correspondence of characteristic peaks in the chromatograms of the test solution and the control medicinal material solution, and then further detecting the citrus aurantium formula granules according to the peak area ratio α1 of rutin naringin to naringin and the peak area ratio α2 of neohesperidin to hesperidin. The UPLC detection method has good accuracy, repeatability and separation, shortens the detection time, and is easy to operate. The present invention uses the simulated formula granule extraction solution as the evaluation benchmark, can accurately detect whether the formula granules are orange and citrus aurantium formula granules, makes up for the vacancy in the technical field of its origin identification, and the method can be used as an exclusive identification method for orange and citrus aurantium formula granules.

Description

Technical Field

[0001] The invention belongs to the technical field of detection of traditional Chinese medicine formula granules, and in particular relates to a detection method for orange and bitter orange formula granules. Background Art

[0002] Citrus aurantium is the dried young fruit of Citrus aurantium L. and its cultivars or Citrus sinensis Osbeck, plants of the Rutaceae family. It is mainly produced in Jiangxi, Sichuan, Fujian, Jiangsu and other places. According to research, Citrus aurantium was first recorded in Shennong's Herbal Classic and listed as a medium-grade product. It has the effects of breaking up qi and eliminating accumulation, resolving phlegm and dispersing lumps, and is a commonly used drug in clinical Chinese medicine. The Citrus aurantium used before the Song Dynasty generally came from the plant Citrus genus of the Rutaceae family. After the Song Dynasty, there were two sources, but the sour orange was the authentic medicinal material. The "upturned belly like the lip of a basin" is the characteristic of the sour orange. Since the Ming and Qing Dynasties, the sour orange has been the authentic source of Citrus aurantium, and the Citrus citrus is second. Sweet orange was first included as a source of Citrus aurantium in the 1995 edition of the Pharmacopoeia of the People's Republic of China. Since sweet orange and sour orange have different origins, their origins, growing conditions and harvesting times are different, resulting in large differences in the chemical composition and content of Citrus aurantium from sour orange and sweet orange, and sour orange is the main one in clinical use. The properties of medicinal materials and decoction pieces under Citrus aurantium in the 2020 edition of the Pharmacopoeia of the People's Republic of China do not distinguish between the appearance characteristics of sour orange and sweet orange, and there are no parameter differences in the other test items.

[0003] Existing technologies have conducted some research on the medicinal material Citrus aurantium. For example, in the article "Study on the Evaluation of Quality Differences of Citrus aurantium Medicinal Materials of Different Origins" published in the 53(14) issue of Chinese Herbal Medicine in 2022, 21 characteristic peaks were selected for quality evaluation, but more than half of the characteristic peaks had too small an area or poor separation. "Study on the Differences in HPLC Fingerprints and Component Contents of Different Varieties of Citrus aurantium" in the 17th issue of "China Journal of Traditional Chinese Medicine" in 2016 recorded a study on fingerprints and component contents using high performance liquid chromatography. The fingerprint of Citrus aurantium dulcis identified 21 common peaks and 12 chromatographic peaks, while the fingerprint of Citrus aurantium dulcis identified 22 common peaks and 11 chromatographic peaks, and their contents were determined, but the separation of some chromatographic peaks was poor; "High Performance Liquid Chromatographic Fingerprint of Citrus aurantium dulcis" in the 9th issue of "Chinese Herbal Medicine" in 2009 recorded the application of high performance liquid chromatography to study the fingerprint of Citrus aurantium dulcis, using a single wavelength and less information on chromatographic peaks. Chinese patent 201610138007.9 (CN106525988A) involves a detection method and standard fingerprint of Zhishi medicinal material, which uses the conversion wavelength method for detection, and the detection wavelength is 280nm and 215nm. The experiment found that when using the 215nm wavelength for detection, due to near-terminal absorption, impurity interference and baseline fluctuation are large. The above studies are all based on Zhishi medicinal materials, and no investigation is conducted on the projects related to Zhishi formula granules. Formula granules are obtained by extracting and concentrating the water-soluble components of medicinal materials. Affected by the extraction efficiency, they are different from the components in the corresponding medicinal materials. At present, there is no public report on the detection method for the identification of the origin of Zhishi formula granules.

[0004] The pilot work of Chinese herbal formula granules in my country ended on November 1, 2021. The main technical reference for standard research and production at this stage is the "Technical Requirements for Quality Control and Standardization of Chinese Herbal Formula Granules" issued by the State Food and Drug Administration, which requires fixed origins for raw materials, that is, different plant or animal sources of the same medicinal material cannot be mixed. After the medicinal materials are processed into decoction pieces and then made into Chinese herbal formula granules, they lose their identification characteristics such as appearance. The extraction, concentration, drying, and granulation of industrial production lead to changes in chemical composition, which increases the difficulty of origin identification. In current clinical use, there are differences in the evaluation of some indications between Citrus aurantium (sour orange) and Citrus aurantium (sweet orange), with Citrus aurantium (sour orange) having more significant effects. In the current national standard for Citrus aurantium (sour orange) formula granules, the characteristic spectrum item contains 6 characteristic peaks. According to the actual measurement of the samples, it is impossible to effectively distinguish between the formula granules with sour orange and sweet orange origins. Therefore, there is currently a lack of a simple and intuitive method for detecting and distinguishing the formula granules of Citrus aurantium (sour orange) and Citrus aurantium (sweet orange). Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect that the existing method cannot distinguish between the granules of Citrus aurantium (sour orange) and the granules of Citrus aurantium (sweet orange), thereby providing a method for identifying the origin of the granules of Citrus aurantium.

[0006] In order to solve the above technical problems, the present invention provides a method for detecting orange and bitter orange formula granules, comprising the following steps:

[0007] (1) Preparation of reference solution:

[0008] Preparation of a reference medicinal material reference solution: taking a reference medicinal material, adding water to boil, filtering to obtain a filtrate, recovering the solvent to obtain a residue, then adding methanol, ultrasonicating, and filtering to obtain the reference medicinal material reference solution;

[0009] Reference substance solution: Take hesperidin reference substance and nobiletin reference substance and add methanol to prepare reference substance solution;

[0010] (2) Preparation of the test solution: Take the Citrus aurantium immaturus granules, grind them, take a sample, accurately weigh them, add methanol, sonicate them, and filter them to obtain the test solution;

[0011] (3) Preparation of simulated granule formula granule solution: taking Citrus aurantium and Citrus aurantium medicinal materials, adding water to decoct, filtering to obtain a filtrate, recovering the solvent to obtain a residue, then adding methanol, ultrasonicating, and filtering to obtain the simulated granule formula granule solution;

[0012] (4) Chromatographic conditions: Determination by ultra-high performance liquid chromatography, with octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution; detection wavelengths at 283 nm and 325 nm; column temperature at 30°C; flow rate at 0.25 ml / min.

[0013] (5) Determination method: Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the ultra-high performance liquid chromatography instrument for determination.

[0014] (6) Establishment of characteristic spectrum: Inject the sample according to the chromatographic conditions of step (4), analyze the reference medicinal material solution, the reference substance solution and the homemade simulated formula granule solution, obtain the corresponding chromatogram, and use the Chinese medicine chromatographic fingerprint similarity evaluation system to generate a fitting graph, thereby determining the standard characteristic spectrum;

[0015] (7) sequentially testing the test sample using the first detection method and the second detection method;

[0016] The first detection method is to compare the ultra-high performance liquid chromatogram of the test sample with the chromatogram of the reference medicinal material solution, and confirm the characteristic peaks using relative retention time and scanning ultraviolet absorption methods;

[0017] The second detection method is to detect based on the peak area ratio α1 of naringin to naringin and the peak area ratio α2 of neohesperidin to hesperidin in the ultra-high performance liquid chromatography of the test solution.

[0018] Furthermore, 11 characteristic peaks should be present in the chromatogram of the test sample, and the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other, and 2 of the peaks should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the hesperidin reference peak is the S1 peak, and the relative retention times of peaks 1, 2, 3, 5, 6, and 7 with the S1 peak should be within ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.89 (peak 2), 0.95 (peak 3), 1.07 (peak 5), peak 6 (1.47), and peak 7 (1.57); the peak corresponding to the nobiletin reference peak is the S2 peak, and the relative retention times of peaks 8, 9, and 11 with the S2 peak should be within ±10% of the specified value. Within the range, the specified values ​​are: 0.89 (peak 8), 0.93 (peak 9), 1.08 (peak 11); scan the absorption spectrum of each characteristic peak in the range of 240-340nm, peaks 1 to 6 should have peak absorption at 283±2nm, peak 7 should have peak absorption at 289±2nm, peak 8 should have peak absorption at 324±2nm, peak 9 should have peak absorption at 322±2nm, peak 10 should have peak absorption at 334±2nm, peak 11 should have peak absorption at 325±2nm.

[0019] Furthermore, the gradient elution procedure is 0-20 min, 12→28% mobile phase A, 88→72% mobile phase B; 20-28 min, 28→47% mobile phase A, 72→53% mobile phase B; 28-37 min, 47% mobile phase A, 53% mobile phase B.

[0020] Furthermore, a wavelength conversion method is adopted, and the detection wavelength is: 283 nm for 0 to 26 min; 325 nm for 26 to 37 min.

[0021] Furthermore, the reference solution is specifically: a mixed solution containing 200 μg of hesperidin and 20 μg of nobiletin per 1 ml.

[0022] Further, the second detection method includes using formula I for detection, when α1 is greater than 5.0 and α2 is greater than 3.0, the test sample formula particles are orange formula particles;

[0023]

[0024] Among them, A2 is the peak area of ​​naringin in the test sample formula granules, A3 is the peak area of ​​naringin in the test sample formula granules, A4 is the peak area of ​​hesperidin in the test sample formula granules, and A5 is the peak area of ​​neohesperidin in the test sample formula granules.

[0025] Beneficial effects of the present invention:

[0026] 1. The present invention adopts ultra high performance liquid chromatography (UPLC) technology to establish the UPLC control characteristic spectrum of the Citrus aurantium (lime) formula granules, determine 11 common peaks, and identify 7 of them. The chromatogram has a stable baseline, good separation of each characteristic peak, and is easy to inspect. It can fully reflect the characteristic peak information of the Citrus aurantium (lime) formula granules, and the method is stable, with high precision, good reproducibility and durability.

[0027] 2. The detection method of the Zhishi (sour orange) formula granules provided by the present invention comprises first comparing the chromatograms of the test solution and the control medicinal material solution, using the relative retention time and scanning ultraviolet absorption method to confirm the characteristic peaks; further analyzing according to the peak area ratio α1 of naringin and rutin naringin, and the peak area ratio α2 of neohesperidin and hesperidin, and controlling the research raw materials by properties on the basis of meeting the relevant batch number requirements of the national "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", ensuring that the original judgment of the research raw materials is accurate, and the corresponding research data and conclusions can effectively distinguish Zhishi (sour orange) from Zhishi (sweet orange) formula granules. The present invention uses the control medicinal material solution as the evaluation benchmark, and the processing method of the research sample is basically the same as the decoction and formula granule industrial extraction method in clinical application. Through multiple limited parameters, it can accurately detect whether the Zhishi formula granules are Zhishi (sour orange) formula granules, which has a high practical application value.

[0028] 3. The detection conditions of the 11 characteristic peaks and the limited values ​​of the two relative peak area ratios used in the present invention are applicable to all the finished products of the Citrus aurantium (sour orange) medicinal material decoctions and formula granules whose origin has been determined to be correct; in some of the Citrus aurantium (sweet orange) medicinal material decoctions and other Citrus aurantium (sweet orange) formula granules with clear origins studied, some characteristic peaks were less than the detection limit requirements (not detected), and all batches of samples could not meet the limited conditions of the two relative peak area ratios. According to the method described in the present invention, they were all judged as non-Citrus aurantium (sour orange) samples or formula granules, and the judgment accuracy was high.

[0029] 4. The conversion wavelength in the characteristic spectrum detection method established by the present invention can more comprehensively reflect the material basis information than a single wavelength, and can more comprehensively reflect the overall quality of the Zhishi (lime) formula granules than the statutory standards. By switching the wavelength, the visual comparison of the characteristic peaks is enhanced; by identifying the characteristic peaks by combining the relative retention time with the maximum ultraviolet absorption, it can effectively avoid the situation where multiple peaks of some characteristic peaks cannot be effectively identified within the specified time window; 7 of the 11 characteristic peaks were identified as components, which more effectively characterized the material basis and quality information of the Zhishi (lime) formula granules, and made up for the defect of determining the origin of Zhishi (lime) after the properties and microscopic identification characteristics were lost after the formula granules were made.

[0030] 5. The method for detecting the Citrus aurantium (lime) formula granules provided by the present invention uses acetonitrile-0.1% formic acid solution as the mobile phase, and adopts gradient and liquid chromatography analysis combined with ultraviolet detector detection to obtain the characteristic spectrum of the Citrus aurantium (lime) formula granules. The origin of the Citrus aurantium formula granules can be determined according to the peak area ratio. The operation is simple and efficient, which fills the gap in the technical field of origin identification of Citrus aurantium (lime) formula granules. This method can be used as an exclusive origin judgment method for Citrus aurantium (lime) formula granules.

[0031] 6. The detection method of the Zhishi (lime) formula granules provided by the present invention, the selected sample pretreatment and instrument detection are all subject to methodological investigation and verification, including sampling volume, solvent investigation, filtration temperature, etc., which is a simulation of the preparation of decoctions and finished products under laboratory conditions, and the preparation process of the formula granules is restored as much as possible, and the degree of reduction is higher than that of the formula granules. The control medicinal material reference solution is extracted by water reflux, and the water extract is evaporated to dryness and then prepared by methanol ultrasonic extraction. The test solution is prepared by methanol ultrasonic method, and the control medicinal material reference chromatogram obtained thereby is more accurate and more specific when compared with the test sample chromatogram. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The liquid chromatogram of the reference solution of the bitter orange (sour orange) medicinal material in Example 1 of the present invention;

[0033] Figure 2 This is a superimposed liquid chromatogram of the simulated formula granules of Citrus aurantium (sour orange) in Example 1 of the present invention;

[0034] Figure 3 This is the fitted liquid chromatogram of the simulated formula granules of Citrus aurantium (sour orange) in Example 1 of the present invention;

[0035] Figure 4 This is a typical characteristic spectrum of the Zhishi (lime) formula granules in Example 1 of the present invention;

[0036] Figures 5-6This is a sample spectrum of non-Fructus Aurantii Immaturus (citrus aurantium) formula granules in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of this application clearer, the technical solution in the embodiment will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the following embodiments are part of the embodiments of this application, but not all of them. Therefore, the following detailed description of the embodiments does not limit the scope of protection claimed in this application. Equivalent replacements or modifications made by ordinary technicians in this field without creative work should be included in the protection scope of the present invention.

[0038] Example 1

[0039] Establishment of standard characteristic spectrum of Zhishi (lime) formula granules

[0040] 1. Instruments and reagents

[0041] 1.1 Instrument

[0042] Instruments: Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph, Agilent 1290 ultra-high performance liquid chromatograph, both equipped with diode array detector; Sartorius CP225D electronic analytical balance; KQ-500DE digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); temperature-controlled electric heating mantle (Shanghai Shuli Instrument Co., Ltd.).

[0043] Column: Thermo Acclaim RSLC C 18 , 2.1mm×100mm, 2.2μm; Waters BEH C 18 , 2.1mm×100mm, 1.8μm; Waters HSS C 18 , 2.1mm×100mm, 1.8μm.

[0044] 1.2 Drug testing

[0045] Methanol, acetonitrile and formic acid were of chromatographic grade, water was purified water, and the other reagents were of analytical grade.

[0046] Reference substances, reference medicinal materials, and sources of medicinal materials are as follows:

[0047] Hesperidin (batch number: 110721-202019, purity: 95.3%, China Food and Drug Inspection Institute); Nobiletin (batch number: 112055-202001, purity: 99.6%, China Food and Drug Inspection Institute); Neohesperidin (batch number: 111857-201804, purity: 99.4%, China Food and Drug Inspection Institute); Tangeretin (batch number: 112054-202001, purity: 99.8%, China Food and Drug Inspection Institute); Citrus citrinin (batch number: U0530010, purity: 98.4 %, Shanghai Anpu Experimental Technology Co., Ltd.); naringin (batch number: 110722-2017114, purity: 93.4%, China Food and Drug Administration); sweet orange flavonoids (batch number: MUST-22011211, purity: 99.64%, Chengdu Mansite Biotechnology Co., Ltd.); rutin (batch number: MUST-22040211, purity: 98.76%, Chengdu Mansite Biotechnology Co., Ltd.); Citrus aurantium (sour orange) reference medicinal material (batch number: 120936-201606, China Food and Drug Administration);

[0048] 16 batches of Citrus aurantium (sour orange) from different production areas, including Yuanjiang, Hunan, Xingan, Jiangxi, Suining, Sichuan, etc. See Table 1.

[0049] Table 1 Information on samples of Citrus aurantium (sour orange)

[0050]

[0051] 2 Methods and Results

[0052] 2.1 Chromatographic conditions and system suitability test: Ultra-high performance liquid chromatography was used as filler, using octadecylsilane bonded silica gel (Thermo Acclaim RSLC C 18 , 2.1mm×100mm, 2.2μm); acetonitrile was used as mobile phase A, 0.1% formic acid solution was used as mobile phase B, and the determination was carried out according to the gradient elution program shown in Table 2; the detection wavelength was 283nm for 0-26min and 325nm for 26-37min; the column temperature was 30°C; the flow rate was 0.25ml / min; the theoretical plate number was calculated based on the hesperidin peak and should be no less than 5000.

[0053] Table 2 Elution gradient

[0054]

[0055] 2.2 Preparation of reference solution, simulated formula particle solution and test solution

[0056] Preparation of reference medicinal material reference solution: Take 0.2g of sour orange reference medicinal material, add 25ml of water, heat and reflux for 30min, filter while hot, evaporate the filtrate to dryness, dissolve the residue with methanol, transfer to a 25ml volumetric flask, ultrasonically treat for 30 minutes, dilute to the mark with methanol, shake well, filter, and take the filtrate as the reference medicinal material solution;

[0057] Take hesperidin reference substance and nobiletin reference substance, add methanol to prepare a mixed solution containing 200 μg of hesperidin and 20 μg of nobiletin per 1 ml, which is used as the reference substance solution.

[0058] Preparation of the test solution: Take the Zhishi formula granules, grind them into powder, take 0.2g, accurately weigh, put it in a stoppered conical flask, accurately add 25ml of methanol, ultrasonically treat for 30 minutes, shake well, filter, and take the filtrate as the test solution.

[0059] Preparation of simulated granule formula solution: Take 0.2g of Citrus aurantium (sour orange) medicinal material, add 25ml of water, heat and reflux for 30min, filter while hot, evaporate the filtrate to dryness, dissolve the residue with methanol, transfer to a 25ml volumetric flask, ultrasonically treat for 30min, dilute to the mark with methanol, shake well, filter, and take the filtrate as the simulated granule formula solution;

[0060] 2.3 Determination method: Accurately pipette 1 μl of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography, and determine.

[0061] 2.4 Determine the detection wavelength

[0062] The above-mentioned sour orange control medicinal material reference solution is taken, and according to the detection conditions of step (1), a diode array detector is used to monitor at a wavelength of 240 to 340 nm, and a chromatogram is recorded. Each target peak is extracted to check the ultraviolet absorption. Analysis shows that dihydroflavonoid components such as naringin, naringin, hesperidin, neohesperidin, and citrus glycosides all have peak absorption near 283 nm, and polymethyl flavonoid components such as nobiletin and tangeretin mostly have peak absorption near 325 nm. In order to more comprehensively reflect the characteristic components in the Zhishi formula granules, while meeting the degree of identification of the characteristic peaks and avoiding the interference of the miscellaneous peaks caused by the terminal absorption, the conversion wavelength method is used for determination, 0 to 26 min is 283 nm; 26 to 37 min is 325 nm.

[0063] 2.5 Determine characteristic peaks

[0064] Establishment of a reference characteristic spectrum of a sour orange medicinal material: according to the preparation method of the reference solution in step (1), 16 batches of sour orange standard medicinal material reference solutions are prepared, and the detection is carried out according to the detection conditions in step (2), and the chromatograms are recorded; the common peak information of the 16 batches of sour orange medicinal materials is analyzed, and the liquid phase spectra of 16 batches of standard Citrus aurantium (citrus) medicinal material extract samples are synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) to establish a reference characteristic spectrum of Citrus aurantium (citrus) medicinal material, and the established characteristic spectrum detection method can be used as a method for determining the origin of Citrus aurantium (citrus) formula granules.

[0065] Through software matching, 11 common peaks were determined in the sour orange medicinal material, and the characteristic spectrum standard of Citrus aurantium (sour orange) was determined as follows: 11 characteristic peaks should be present in the chromatogram of the test sample, and correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material, of which 2 peaks correspond to the retention times of the corresponding reference peaks. The peak corresponding to the hesperidin reference peak is the S1 peak, and the relative retention times of peaks 1, 2, 3, 5, 6, and 7 with S1 are calculated, which should be within the range of ±10% of the specified value, and the specified values ​​are: 0.77 (peak 1), 0.89 (peak 2), 0.95 (peak 3), 1.07 (peak 5), 1.47 (peak 6), and 1.57 (peak 7); the peak corresponding to the nobiletin reference peak is the S2 peak, and the relative retention times of peaks 8, 9, and 11 with S2 are calculated, which should be within the range of ±10% of the specified value. Within the range, the specified values ​​are: 0.89 (peak 8), 0.93 (peak 9), 1.08 (peak 11); scan the absorption spectrum of each characteristic peak in the range of 240-340nm, peaks 1 to 6 should have peak absorption at 283±2nm, peak 7 should have peak absorption at 289±2nm, peak 8 should have peak absorption at 324±2nm, peak 9 should have peak absorption at 322±2nm, peak 10 should have peak absorption at 334±2nm, peak 11 should have peak absorption at 325±2nm.

[0066] Confirmation of chromatographic peaks: According to the identification of characteristic peaks based on the reference substance, peak 2 is naringin, peak 3 is naringin, peak 4 is hesperidin, peak 5 is neohesperidin, peak 6 is citrus glycoside, peak 10 is nobiletin, and peak 11 is tangeretin.

[0067] Table 3 Summary of relative retention times of 16 batches of simulated formula granules of Citrus aurantium (lime)

[0068]

[0069] Table 4 Relative peak areas of 16 batches of simulated formula granules of Citrus aurantium (lime)

[0070]

[0071]

[0072] 2.6 Detection method: The test solution is tested using the first detection method and the second detection method in sequence;

[0073] The first detection method is to compare the characteristic peaks in the chromatogram of the test sample with the characteristic peaks in the chromatogram of the reference medicinal material to obtain determination result 1. If the relevant requirements of determination principle 1 are met, continue to detect according to the second method;

[0074] Analysis of the chromatogram results of the test sample. In the figure, 11 characteristic peaks corresponding to the reference medicinal materials are presented in the chromatogram of the test sample, of which 2 peaks correspond to the retention times of the corresponding reference peaks of the reference substance, and the peak corresponding to the hesperidin reference peak is the S1 peak. The relative retention times of peaks 1, 2, 3, 5, 6, and 7 with S1 are calculated to be within the range of ±10% of the specified values, and the specified values ​​are: 0.77 (peak 1), 0.89 (peak 2), 0.95 (peak 3), 1.07 (peak 5), 1.47 (peak 6), and 1.57 (peak 7); the peak corresponding to the nobiletin reference peak is the S2 peak, and the retention time of peak 1, peak 2, peak 3, peak 5, peak 6, and peak 7 with S1 is ...). The relative retention time of peak 8, peak 9, peak 11 and S2 should be within the range of ±10% of the specified value, and the specified value is: 0.89 (peak 8), 0.93 (peak 9), 1.08 (peak 11); scanning the absorption spectrum of each characteristic peak in the range of 240-340nm, peak 1-peak 6 should have peak absorption at 283±2nm, peak 7 should have peak absorption at 289±2nm, peak 8 should have peak absorption at 324±2nm, peak 9 should have peak absorption at 322±2nm, peak 10 should have peak absorption at 334±2nm, and peak 11 should have peak absorption at 325±2nm. If the above conditions are met, the test sample (formulated granules) is considered to be the formula granules of Citrus aurantium (lime).

[0075] The second detection method is: use formula Ⅰ for detection,

[0076]

[0077] Wherein, A2 is the peak area of ​​naringin in the test sample granules, A3 is the peak area of ​​naringin in the test sample granules, A4 is the peak area of ​​hesperidin in the test sample granules, and A5 is the peak area of ​​neohesperidin in the test sample granules. When α1 is greater than 5.0 and α2 is greater than 3.0, the test sample granules are Citrus aurantium (sour orange) granules;

[0078] The peak areas of Peak 2, Peak 3, Peak 4 and Peak 5 and the values ​​of α1 and α2 in the chromatograms of the standard medicinal materials and test samples are shown in the table.

[0079] Table 5 Peak area determination data of 6 batches of formula granules determined to be derived from Citrus aurantium (lime)

[0080] No. / Peak Area <![CDATA[A2]]> <![CDATA[A3]]> <![CDATA[A4]]> <![CDATA[A5]]> <![CDATA[α1(A3 / A2)]]> <![CDATA[α2(A5 / A4)]]> Test sample 1 1070388 11032306 700624 8237972 10.307 11.758 Test sample 2 1064794 10981908 699326 8192980 10.314 11.716 Test sample 3 498141 9682861 1009751 12415046 19.438 12.295 Test sample 4 616520 11883925 1271317 15249836 19.276 11.995 Test sample 5 490007 9505204 1009811 12193989 19.398 12.076 Test sample 6 583019 8635076 617174 10797743 14.811 17.495

[0081] For the data listed in the above table, the α1 of the test sample is greater than 5.0 and the α2 is greater than 3.0. The test results of the test sample are the same as the actual feed according to the proposed judgment conditions, and the origin is Citrus aurantium (orange).

[0082] By combining the first detection method and the second detection method, it was confirmed that the test sample formula granules were the Zhishi (lime) formula granules.

[0083] 2.4 Methodological Review

[0084] 2.4.1 Precision test

[0085] Take the sour orange formula granules, prepare the test solution according to the method of step (2) of Example 1, inject 6 times continuously, 1 μL each time, detect according to the chromatographic conditions of step (1) of Example 1, record the chromatographic peaks, find the common characteristic peaks, and obtain the characteristic spectrum. Take peak No. 4 as the reference peak S1 peak and peak No. 10 as the reference peak S2 peak, calculate the relative peak area and relative retention time of the remaining characteristic peaks, and their RSD values ​​are all less than 1.0%, indicating that the precision of the instrument is good.

[0086] 2.4.2 Repeatability test

[0087] Take 6 portions of the orange formula granules (samples), prepare a test solution according to the method of step (2) of Example 1, and detect according to the chromatographic conditions of step (1) of Example 1, record the chromatographic peaks, use peak 4 as the reference peak S1 and peak 10 as the reference peak S2, calculate the relative retention time and relative peak area of ​​the remaining characteristic peaks, and their RSD values ​​are all less than 1.0%, indicating that the method has good repeatability.

[0088] 2.4.3 Stability test

[0089] Take the sour orange formula granules (sample), prepare the test solution according to the method of step (2) of Example 1, detect at the specified time point within 24 hours according to the chromatographic conditions of step (1) of Example 1, record the chromatographic peaks, use peak No. 4 as the reference peak S1 peak and peak No. 10 as the reference peak S2 peak, calculate the relative peak area and relative retention time of the remaining characteristic peaks, and calculate the RSD value. The relative retention time and relative peak area RSD values ​​of each characteristic peak are all less than 1.0%, indicating that the test component in the test solution has good stability within 24 hours under the present extraction and determination conditions.

[0090] 2.4.4 Durability test

[0091] Using 2 different brands of ultra-high performance liquid chromatographs and 3 C 18The chromatographic columns were inspected. Chromatographs: Waters UPLCH-Class, Agilent 1290; chromatographic columns: Thermo Acclaim RSLC, 2.1mm×100mm, 2.2μm, WatersBEH, 2.1mm×100mm, 1.8μm, Waters HSS T3, 2.1mm×100mm, 1.8μm. The results of the relative retention time, UV maximum spectrum absorption and relative peak area were consistent, and the 11 characteristic peaks could be effectively detected and met the corresponding requirements, indicating that the chromatographic column of the method had good durability.

[0092] Table 6 Summary of relative retention time of durability test

[0093]

[0094] Table 7 Summary of maximum absorption wavelengths in durability tests (unit: nm)

[0095]

[0096] Table 8 Summary of relative peak areas in durability tests

[0097]

[0098] Determination of characteristic spectra of Zhishi (lime) formula granules

[0099] 1.1 Instrument

[0100] Instruments: Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph, equipped with a diode array detector, Empower 3 chromatography workstation; Sartorius CP225D electronic analytical balance; KQ-500DE digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). The chromatographic column was Thermo Acclaim RSLC C 18 , 2.1mm×100mm, 2.2μm.

[0101] 1.2 Drug testing

[0102] Methanol, acetonitrile and formic acid were of chromatographic grade, water was purified water, and the other reagents were of analytical grade.

[0103] Reference substances, reference medicinal materials, and sources of medicinal materials are as follows:

[0104] Hesperidin (batch number: 110721-202019, purity: 95.3%, China Food and Drug Administration); Nobiletin (batch number: 112055-202001, purity: 99.6%, China Food and Drug Administration);

[0105] 19 batches of Citrus aurantium formula granules samples were randomly collected during the production and circulation stages. The sample information is shown in the attached table.

[0106] The test was carried out according to the method of the present invention, and the test results are shown in the following table:

[0107] It was detected that 8 batches had obvious missing common peaks (peak 1, peak 3, peak 5, peak 7, peak 8, peak 9) compared with the characteristic spectrum of the control of Zhishi (lime), of which 4 batches did not match the actual identification base (identified as Zhishi (lime) formula granules), and the other 4 batches did not identify the base (identified as Zhishi formula granules); 5 batches were detected with 11 common peaks, but the relative peak area ratio α1 was less than 5.0, and α2 was less than 3.0, which was speculated to be mixed with multiple bases; 6 batches met the judgment basis of this method for Zhishi (lime) formula granules. Therefore, by constructing the UPLC characteristic spectrum, it is possible to effectively identify whether the Zhishi formula granules are the base of lime, which is helpful for evaluating the quality of Zhishi formula and assisting clinical medication.

[0108] Determination of sweet orange impersonation or mixing

[0109] When sweet orange is used as sour orange, some characteristic peaks in this method may not be effectively detected; if interference peaks appear within the relative retention time range and cannot be judged due to the complexity of the Chinese medicine ingredients, the relative peak area ratio can be used to make a judgment.

[0110] If sweet orange is mixed with sour orange, since sweet orange has high content of naringin and hesperidin, and basically does not contain naringin and neohesperidin, although this method can effectively detect the specified characteristic peaks when applied, the relative peak area ratio will be greatly affected. The mixing or adulteration caused by incorrect judgment of the origin of the raw materials will result in a relative peak area ratio exceeding the specified range, and corresponding judgment can also be made.

[0111] Table 9 Measurement results of commercially available samples

[0112] serial number Manufacturer batch number Packaging origin identification Gene detection results 1 A 2010001 lime ×(characteristic peak missing) 2 A 2010002 lime ×(characteristic peak missing) 3 A 2010003 lime ×(characteristic peak missing) 4 B 21041415 Sweet Orange ×(Lime) 5 B 21070082 Sweet Orange ×(Lime) 6 B 2010041 lime √(Lime) 7 B 2010042 lime √(Lime) 8 B 2010043 lime √(Lime) 9 C 22003071 lime √(Lime) 10 D 1903001 Unidentified Non-orange (characteristic peak missing) 11 D 1905001 Unidentified Non-acid orange (relative peak area ratio exceeds the specified value) 12 D 1908001 Unidentified Non-acid orange (relative peak area ratio exceeds the specified value) 13 D 1910001 Unidentified Non-acid orange (relative peak area ratio exceeds the specified value) 14 D 1911001 Unidentified Non-orange (characteristic peak missing) 15 D 2001001 Unidentified Non-orange (characteristic peak missing) 16 D 2102001 Unidentified Non-acid orange (relative peak area ratio exceeds the specified value) 17 D 2108001 Unidentified Non-acid orange (relative peak area ratio exceeds the specified value) 18 D 2109001 Unidentified Non-orange (characteristic peak missing) 19 D 2112001 lime ×(characteristic peak missing)

[0113] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for detecting Citrus aurantium and Citrus aurantium formula granules, characterized in that: The steps include: (1) Preparation of reference solution: Preparation of a reference medicinal material reference solution: taking a reference medicinal material, adding water to boil, filtering to obtain a filtrate, recovering the solvent to obtain a residue, then adding methanol, ultrasonicating, and filtering to obtain the reference medicinal material reference solution; Preparation of reference substance solution: Take hesperidin reference substance and nobiletin reference substance, weigh accurately, and add methanol to prepare reference substance solution; (2) Preparation of the test solution: Take the Zhishi formula granules, grind them, take a sample, accurately weigh them, add methanol, sonicate them, and filter them to obtain the test solution; (3) Preparation of simulated formula granule solution: taking Citrus aurantium and Citrus aurantium medicinal materials, adding water to decoct, filtering to obtain a filtrate, recovering the solvent to obtain a residue, then adding methanol, ultrasonicating, and filtering to obtain the simulated formula granule solution; (4) Chromatographic conditions: ultra-high performance liquid chromatography, octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution; diode array detector, detection wavelength 283 nm from 0 to 26 min, 325 nm from 26 to 37 min, full wavelength scan in the range of 240 to 340 nm; column temperature 30°C; flow rate 0.25 ml / min; (5) Determination method: Accurately pipette 1 μL of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine; (6) Establishment of characteristic spectrum: Inject the sample according to the chromatographic conditions of step (4), analyze the reference medicinal material solution, the reference substance solution and the homemade simulated formula granule solution, obtain the corresponding chromatogram, and use the Chinese medicine chromatographic fingerprint similarity evaluation system to generate a fitting graph, thereby determining the standard characteristic spectrum; (7) Determination method: The test solution is tested using the first detection method and the second detection method in sequence; The first detection method is to detect the liquid chromatography of the test solution and the liquid chromatography of the control medicinal material reference solution and the control substance reference solution, wherein the 9 peaks are located using relative retention time combined with the maximum absorption wavelength of the characteristic peak; The second detection method is to detect based on the peak area ratio ɑ1 of naringin and naringin, and the peak area ratio ɑ2 of neohesperidin and hesperidin in the chromatogram of the test solution; The chromatogram of the sour orange and bitter orange formula granules presents 11 characteristic peaks, and the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material reference substance correspond to each other, wherein 2 peaks should correspond to the retention times of the corresponding reference substance peaks respectively; the peak corresponding to the hesperidin reference substance peak is the S1 peak, and the relative retention times of peaks 1, 2, 3, 5, 6, 7 and the S1 peak are calculated to be within ±10% of the specified value, and the specified values ​​are: peak 1: 0.77, peak 2: 0.89, peak 3: 0.95, peak 5: 1.07, peak 6: 1.47, peak 7: 1.57; the relative retention times of the nobiletin reference substance peaks are: The corresponding peak is the S2 peak, and the relative retention time of peaks 8, 9, 11 and S2 peak is calculated, which is within the range of ±10% of the specified value, and the specified value is: peak 8: 0.89, peak 9: 0.93, peak 11: 1.08; the absorption spectrum of each characteristic peak in the range of 240-340nm is scanned, and peaks 1-6 have peak absorption at 283±2nm, peak 7 has peak absorption at 289±2nm, peak 8 has peak absorption at 324±2nm, peak 9 has peak absorption at 322±2nm, peak 10 has peak absorption at 334±2nm, and peak 11 has peak absorption at 325±2nm; The gradient elution procedure is 0-20 min, 12→28% mobile phase A, 88→72% mobile phase B; 20-28 min, 28→47% mobile phase A, 72→53% mobile phase B; 28-37 min, 47% mobile phase A, 53% mobile phase B; The wavelength conversion method is adopted, and the detection wavelength is: the detection wavelength of peak 1 to peak 7 is 283nm at 0 to 26min; the detection wavelength of peak 8 to peak 11 is 325nm at 26 to 37min; The reference solution is specifically: a mixed solution containing 200 μg of hesperidin and 20 μg of nobiletin per 1 ml; The second detection method includes using formula I for detection, when ɑ1 is greater than 5.0 and ɑ2 is greater than 3.0, the test sample formula granules are orange and bitter orange formula granules; Among them, A2 is the peak area of ​​naringin in the formula granule test sample, A3 is the peak area of ​​naringin in the formula granule test sample, A4 is the peak area of ​​hesperidin in the formula granule test sample, and A5 is the peak area of ​​neohesperidin in the formula granule test sample.

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