Quantitative analysis method of multi-component simultaneous determination in pericarpium citri reticulatae

Abstract

The application provides a quantitative analysis method for multi-component quantitative analysis of pericarpium citri reticulatae, and belongs to the technical field of pericarpium citri reticulatae detection.The quantitative analysis method comprises the following steps: preparing mixed control samples of naringin, hesperidin, citropten and tangeretin; performing ultrasonic extraction on pericarpium citri reticulatae powder, supplementing the lost solvent by using an extraction system with an automatic liquid supplementing device to obtain a test sample; and performing HPLC determination, taking hesperidin as an internal reference, and calculating the content of each component by using a one-sample-multiple-estimation method.The methodological verification shows that the linearity is good, the recovery rate is 97.59% to 100.68%, and the RSD is less than or equal to 1.92%; the extraction device significantly improves the precision.The application is simple, accurate, and good in repeatability, and is suitable for quality control of pericarpium citri reticulatae and citrus medicinal materials.

Description

Technical Field

[0001] This invention relates to the field of tangerine peel detection technology, and in particular to a quantitative analysis method for multiple components in tangerine peel that can be measured and evaluated in one step. Background Technology

[0002] The current standard for dried tangerine peel is the 2025 edition of the Chinese Pharmacopoeia, which officially came into effect on October 1, 2025. Standards for the content of effective components have been established for both Guangchenpi (Guangdong-style dried tangerine peel) and other dried tangerine peel medicinal materials. The quantitative indicator for dried tangerine peel is hesperidin, while the quantitative indicators for Guangchenpi are hesperidin, nobiletin, and citrus terpenoids. In this standard, the hesperidin content in Guangchenpi is significantly lower than that in other dried tangerine peel medicinal materials, and these materials also contain polymethoxylated flavonoids such as nobiletin and citrus terpenoids. Therefore, it is urgent to analyze the differences in major representative and indicative components between cultivated varieties and medicinal dried tangerine peel, and to explore the feasibility of using cultivated varieties as medicinal dried tangerine peel based on their chemical composition.

[0003] In conventional testing methods, ultrasonic extraction of tangerine peel and similar medicinal materials can lead to inaccurate volume and concentration due to solvent loss. Manual replenishment requires operators to repeatedly observe the liquid level or pipette scale and judge the amount to be added based on experience. This is prone to over- or under-addition due to visual errors or operator fatigue, introducing human bias and reducing experimental repeatability. Summary of the Invention

[0004] The purpose of this invention is to provide a quantitative analysis method for multiple components in dried tangerine peel, which can solve the above-mentioned technical problems.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a quantitative analysis method for multiple components in dried tangerine peel, comprising the following steps: 1) Accurately weigh the naringin standard, hesperidin standard, norihesperidin standard and citrus peel standard, mix them with solvent and dilute them to obtain a series of mixed reference solutions of different concentrations; 2) Accurately weigh the dried tangerine peel powder and place it in an Erlenmeyer flask. Accurately add the extractant, and after ultrasonic extraction, replenishment, shaking and filtration, the test solution is obtained. 3) Inject the series of mixed reference solutions and test solutions separately according to the chromatographic conditions and record the peak area of ​​each component; plot the standard curve with the mass concentration of each reference standard as the abscissa and the peak area as the ordinate, and calculate the regression equation; 4) The method of one test and multiple evaluations was adopted, and hesperidin was used as an internal reference. The content of each analyte was calculated according to the formula of relative correction factor. In step 2), the replenishment and mixing are performed in an extraction device, which includes: The platform is equipped with a constant temperature shaking table; A clamp is disposed inside the constant temperature shaker and is used to fix the conical flask; An electronic scale is installed on the platform and located on one side of the constant temperature shaker. A tray is fixedly installed on the electronic scale for placing conical flasks to be replenished. A solvent replenishment mechanism, mounted on the platform, is used to replenish the solvent into the conical flask to make up for the lost weight.

[0006] Furthermore, in step 1), the solvent includes methanol or an aqueous methanol solution; In the series of mixed reference solutions, the concentrations of naringin, hesperidin, nobiletin, and citrusin were 3-65 μg / mL, 25-510 μg / mL, 3-65 μg / mL, and 1-35 μg / mL, respectively.

[0007] Furthermore, in step 2), the extractant includes methanol or an aqueous methanol solution; The ratio of tangerine peel powder to extractant is 0.1~0.4g:25~30mL.

[0008] Furthermore, in step 3), the chromatographic conditions are as follows: Chromatographic column: C18 column; Mobile phase: acetonitrile-0.05% phosphoric acid solution; Detection wavelength: 280~330nm; Column temperature: 25~30℃; Flow rate: 0.8~1.2 mL / min; Gradient elution program: 0-15 min, 10% acetonitrile; 15-16 min, 35% acetonitrile; 16-40 min, 40% acetonitrile; 40-46 min, 60% acetonitrile; 46-50 min, 10% acetonitrile.

[0009] Furthermore, in step 4), the formula for calculating the relative correction factor is as follows: , Among them, A i C represents the peak area of ​​a certain analyte i; i Let A be the concentration of the analyte i. s C is the peak area of ​​the internal reference s. s The concentration of the internal reference substance s.

[0010] Furthermore, in the extraction device, the clamp includes a placement plate and a plurality of clamp bodies arranged in an array on the placement plate. The placement plate has a set of grooves, each of which includes a circular groove and a plurality of elongated grooves arranged circumferentially around the circular groove. The side of the placement plate has a slot, which is located below the circular groove and communicates with the circular groove. The inner walls on both sides of the slot have sliding grooves, and a magnet is fixed inside the slot.

[0011] Furthermore, the clamp body includes: Multiple clamping plates are provided, each corresponding to one of the elongated grooves. Each clamping plate is an elastically bent component, comprising a first clamping part and a second clamping part connected at an acute angle, and the connection between the two parts is rotatably mounted on one side edge of the corresponding elongated groove. The first clamping part is used to abut against the outer wall of the conical bottle, and the second clamping part is used to abut against the lower surface of the placement plate in the initial state. The base has an outer contour that matches the circular groove, and the base abuts against the upper surface of each of the second clamping parts; a fixing post is fixedly connected to the center bottom of the base, the fixing post passes through the circular groove and extends into the corresponding slot, and an iron block is fixed to the bottom end of the fixing post, and a wedge-shaped notch is opened at the bottom end of the iron block; A return spring is sleeved on the outer periphery of the fixed column and elastically supported between the bottom wall of the circular groove and the lower surface of the base. A wedge-shaped insert plate, wherein both sides of the wedge-shaped insert plate are provided with protrusions that slide in conjunction with the groove; When the wedge-shaped insert is pushed into the slot, the wedge-shaped surface of the wedge-shaped insert abuts against the wedge-shaped notch to push the iron block to overcome the attraction of the magnet and separate. At the same time, the return spring releases the elastic force and pushes the fixing column to move upward, so that the base moves upward and each of the clamps rotates outward around its connection to release the conical bottle. Each of the first clamping parts is equipped with a rubber pad.

[0012] Furthermore, the solvent replenishment mechanism includes: A support frame, wherein a liquid outlet is provided on the support frame and the liquid outlet is located directly above the tray; A solvent storage tank is disposed on the side of the bracket away from the electronic scale; A pump is provided on one side of the solvent storage tank, with its inlet end connected to the solvent storage tank and its outlet end connected to the outlet via a pipe. A metering valve, which is installed on the pipeline, is used to control the amount of solvent replenished.

[0013] The beneficial effects of this invention are: This invention employs a single-measurement, multi-evaluation method, using hesperidin as a single internal reference to simultaneously achieve quantitative analysis of multiple components such as naringin, nosocomiotertin, and citrus peel glycosides. This eliminates the need to purchase multiple high-purity reference standards individually, significantly reducing detection costs. It is particularly suitable for scenarios where reference standards are expensive or difficult to obtain.

[0014] This invention achieves good separation of four analytes in a short time through systematic optimization of chromatographic conditions, with symmetrical peaks, stable response, and strong method specificity, effectively eliminating the influence of other interfering components in tangerine peel.

[0015] The quantitative analysis method provided by this invention offers a reliable detection means for comparing the differences in chemical composition between cultivated varieties (such as Ehime 38 and Papa Mandarin Orange) and authentic Chenpi (dried tangerine peel). It helps to assess the feasibility of using cultivated varieties as medicinal Chenpi from a chemical perspective, and has practical guiding significance for the resource expansion and quality control of Chenpi medicinal materials. It is easy to promote and apply in routine quality inspection laboratories and is suitable as a general method for multi-index quality evaluation of Chenpi and citrus medicinal materials.

[0016] This invention uses an electronic scale, a tray, and a solvent replenishment mechanism to work together. The electronic scale can weigh the conical flask to be replenished in real time and feed back the weight loss to the metering valve. The pump accurately injects the solvent from the solvent storage tank into the conical flask through the outlet, avoiding visual errors and operational deviations caused by manual liquid handling.

[0017] The clamp of this invention adopts a linkage structure of elastic bending clamps and wedge-shaped inserts. When filling the bottle, simply press the conical bottle and its base together into the circular groove. The iron block is attracted and fixed by the magnet. The first clamping part of each clamp automatically retracts inward and clamps the conical bottle after being squeezed by the base. When releasing, simply push the wedge-shaped insert into the slot. The wedge-shaped surface matches the wedge-shaped notch of the iron block, pushing the iron block to overcome the magnetic attraction. The return spring releases the elastic force to push the base upward, and each clamp rotates outward synchronously to release the conical bottle. This greatly reduces the time spent by the experimenter in the clamping and bottle removal process, and is especially suitable for the processing of batch samples. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the extraction device of the present invention. Figure 1 ; Figure 2 This is a schematic diagram of the extraction device of the present invention. Figure 2 ; Figure 3 This is a schematic diagram of the clamping fixture in the clamping state of an extraction device according to the present invention; Figure 4 This is a schematic diagram of the clamp in the unclamped state of an extraction device according to the present invention; Figure 5 This is a schematic diagram of the wedge-shaped insert structure of an extraction device according to the present invention.

[0019] In the diagram: 1. Platform; 2. Electronic scale; 3. Thermostatic shaker; 4. Placement plate; 5. Circular groove; 6. Long strip groove; 7. Slot; 8. Clamping plate; 9. First clamping part; 10. Second clamping part; 11. Base; 12. Fixing column; 13. Tray; 14. Return spring; 15. Wedge-shaped insert; 16. Slide groove; 17. Protrusion; 18. Bracket; 19. Solvent storage tank; 20. Pump; 21. Pipeline. Detailed Implementation

[0020] This invention provides a quantitative analysis method for multiple components in dried tangerine peel, using a single measurement and evaluation approach. The extraction device is used as follows: a constant-temperature shaker is fixedly installed on one side of a platform, ensuring the shaker base is securely connected to the platform surface with bolts to prevent displacement during oscillation. An electronic scale is installed adjacent to the constant-temperature shaker, with its weighing surface leveled using leveling feet at the bottom. A tray is fixedly installed on the weighing surface of the electronic scale. An anti-slip pad (such as a silicone pad) can be provided on the upper surface of the tray to increase the stability of the conical flask. A support is erected on the platform, with its upper outlet aligned with the center of the tray to ensure accurate solvent dripping into the conical flask during replenishment.

[0021] In practice, the operator first places the bottom of the conical flask on the upper surface of the base, holds the body of the conical flask with his hand and presses it down, so that the base moves down together with the conical flask.

[0022] In the initial state, the connection between the first and second clamping parts of each clamping plate is rotatably mounted on one edge of the elongated groove. The second clamping parts hang naturally under their own weight and abut against the lower surface of the placement plate. When the base moves downwards, the lower surface of the base contacts the upper surface of each second clamping part. Since the second clamping part is connected to the first clamping part at an acute angle, when the second clamping part is pressed down, the first clamping part rotates inwards around its connection point with the edge of the elongated groove, gradually approaching and finally abutting against the outer wall of the conical flask. Simultaneously, the fixing post moves downwards synchronously with the base, passing through the circular groove and extending into the slot. The iron block at the bottom of the fixing post gradually approaches the magnet fixed in the slot. As the pressure continues until the iron block attracts the magnet, the return spring is compressed, the base reaches its lowest position, and the first clamping parts of each clamping plate apply a moderate clamping force to the outer wall of the conical flask under elastic deformation, thereby firmly fixing the conical flask to the placement plate.

[0023] Afterwards, the operator places the entire placement plate inside the constant temperature shaker and starts the shaker to perform oscillation extraction for the set time.

[0024] After the shaking extraction is complete, turn off the constant temperature shaker, open the shaker door, and remove the entire placement plate.

[0025] Finally, the operator aligns the two protrusions on both sides of the wedge-shaped insert with the grooves on the inner walls of the slot and pushes it into the slot. The wedge-shaped surface (i.e., the gradually narrowing slope) of the wedge-shaped insert first contacts the wedge-shaped notch at the bottom of the iron block. As the wedge-shaped insert continues to go deeper, the wedge-shaped surface and the wedge-shaped notch slide relative to each other, applying an upward pushing force to the iron block. This pushing force is used to overcome the magnetic attraction between the iron block and the magnet. Once the magnetic attraction is completely overcome, the iron block separates from the magnet.

[0026] At this point, the return spring is no longer subjected to downward pressure, and its stored elastic potential energy is released, pushing the fixed column upward along the circular groove, and the base moves upward synchronously. After the base moves upward, its lower surface no longer applies pressure to the second clamping parts, and each of the second clamping parts loses its downward constraint. The first clamping part rotates outward, releasing its contact with the outer wall of the conical flask, and the conical flask can be easily removed.

[0027] In this invention, the experimental instruments include: a Shimadzu LC-20A high-performance liquid chromatograph (SPD-M20A photodiode array UV-Vis detector, CTO-20AC column oven, SIL-20ACHT UFLC high-performance liquid chromatograph autosampler); a digital display constant temperature water bath (DZKW-C type, Shanghai Shuli Instrument Co., Ltd.); an analytical balance (XS205DU type, Mettler Toledo International Ltd.); a CNC ultrasonic cleaner (KQ-700DE type, Kunshan Ultrasonic Instrument Co., Ltd.); and an ultrapure water system (UPR-II-20L type, Sichuan Youpu Ultrapure Technology Co., Ltd.).

[0028] In this invention, the experimental reagents and reagents include: naringin reference standard (batch number 1212-RF-0087) purchased from CATO Research Chemicals Inc.; hesperidin (batch number 110721-202220) purchased from the China National Institutes for Food and Drug Control; tangeretin (batch number 15558, NATURE STANDARD); and citrus (batch number 16321, NATURESTANDARD); acetonitrile (chromatographic grade, Chengdu Kelong Chemicals Co., Ltd.); ultrapure water (self-made using a UPU ultrapure water system); and phosphoric acid (chromatographic grade, Chengdu Kelong Chemicals Co., Ltd.).

[0029] In this invention, the medicinal materials used in the experiment were purchased or collected by the research group from the Chengdu Traditional Chinese Medicine Market and different citrus-growing areas in Meishan City between 2024 and 2025, with a total of 16 batches of medicinal materials collected from different producing areas. Table 1 shows the sources of the medicinal materials.

[0030] Table 1. Sources of medicinal materials

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] refer to Figures 1-5 An extraction device, comprising: Platform 1, on which a constant temperature shaking table 3 is installed; The clamp is located inside the constant temperature shaker 3 and is used to fix the conical flask. Electronic scale 2 is installed on platform 1 and located on one side of constant temperature shaker 3. A tray 13 is fixedly installed on electronic scale 2. The tray 13 is used to place conical flasks to be replenished. A solvent replenishment mechanism, installed on platform 1, is used to replenish the solvent in the conical flask to make up for the lost weight.

[0034] In this embodiment, the fixture includes a placement plate 4 and a plurality of fixture bodies arranged in an array on the placement plate 4. The placement plate 4 has a plurality of groove groups, each groove group including a circular groove 5 and a plurality of elongated grooves 6 arranged circumferentially around the circular groove 5. The side of the placement plate 4 has a slot 7, which is located below the circular groove 5 and communicates with the circular groove 5. The inner walls on both sides of the slot 7 are provided with sliding grooves 16, and a magnet is fixed inside the slot 7.

[0035] In this embodiment, the fixture body includes: Multiple clamping plates are provided, and multiple clamping plates are correspondingly arranged with multiple elongated grooves 6. The clamping plate 8 is an elastic bending member, which includes a first clamping part 9 and a second clamping part 10 connected at an acute angle, and the connection between the two parts is rotatably installed on one side edge of the corresponding elongated groove 6. The first clamping part 9 is used to abut against the outer wall of the conical bottle, and the second clamping part 10 is used to abut against the lower surface of the placement plate 4 in the initial state. The base 11 has an outer contour that matches the circular groove 5 and abuts against the upper surface of each second clamping part 10. A fixing post 12 is fixedly connected to the center bottom of the base 11. The fixing post 12 passes through the circular groove 5 and extends into the corresponding slot 7. An iron block is fixed at the bottom end of the fixing post 12, and a wedge-shaped notch is opened at the bottom end of the iron block. Return spring 14 is sleeved on the outer periphery of fixed post 12 and elastically supported between the bottom wall of circular groove 5 and the lower surface of base 11. The wedge-shaped insert plate 15 has protrusions 17 on both sides that slide in conjunction with the slide groove 16. When the wedge-shaped insert 15 is pushed into the slot 7, the wedge-shaped surface of the wedge-shaped insert 15 abuts against the wedge-shaped notch to push the iron block to overcome the attraction of the magnet and separate. At the same time, the return spring 14 releases the elastic force and pushes the fixed column 12 to move upward, so that the base 11 moves upward and each clamp 8 rotates outward around its connection to release the conical bottle.

[0036] In this embodiment, each of the first clamping parts 9 is provided with a rubber pad.

[0037] In this embodiment, the solvent replenishment mechanism includes: The support 18 is provided with a liquid outlet, which is located directly above the tray 13; Solvent storage tank 19 is located on the side of the bracket 18 away from the electronic scale 2; Pump 20 is installed on one side of solvent storage tank 19, and the inlet end of pump 20 is connected to solvent storage tank 19. The outlet end of pump 20 is connected to outlet through pipe 21. A metering valve is installed on pipe 21 to control the amount of solvent replenished.

[0038] In practice, it also includes a controller, which is electrically connected to the metering valve, the pump, and the electronic scale.

[0039] Instructions for use: Securely install the thermostatic shaker to one side of the platform, ensuring the shaker base is firmly bolted to the platform surface to prevent displacement during shaking. Install the electronic scale on the adjacent side of the thermostatic shaker, ensuring the weighing surface of the scale is level. Level the scale using the leveling feet at its bottom. Securely install the tray onto the weighing surface of the electronic scale. A non-slip pad (such as a silicone pad) can be installed on the upper surface of the tray to increase the stability of the conical flask. Erect the support stand on the platform, ensuring the liquid outlet at its top is aligned with the center of the tray to ensure accurate liquid delivery of the solvent solution into the conical flask during replenishment.

[0040] In practice, the operator first places the bottom of the conical flask on the upper surface of the base, holds the body of the conical flask with his hand and presses it down, so that the base moves down together with the conical flask.

[0041] In the initial state, the connection between the first and second clamping parts of each clamping plate is rotatably mounted on one edge of the elongated groove. The second clamping parts hang naturally under their own weight and abut against the lower surface of the placement plate. When the base moves downwards, the lower surface of the base contacts the upper surface of each second clamping part. Since the second clamping part is connected to the first clamping part at an acute angle, when the second clamping part is pressed down, the first clamping part rotates inwards around its connection point with the edge of the elongated groove, gradually approaching and finally abutting against the outer wall of the conical flask. Simultaneously, the fixing post moves downwards synchronously with the base, passing through the circular groove and extending into the slot. The iron block at the bottom of the fixing post gradually approaches the magnet fixed in the slot. As the pressure continues until the iron block attracts the magnet, the return spring is compressed, the base reaches its lowest position, and the first clamping parts of each clamping plate apply a moderate clamping force to the outer wall of the conical flask under elastic deformation, thereby firmly fixing the conical flask to the placement plate.

[0042] Afterwards, the operator places the entire placement plate inside the constant temperature shaker and starts the shaker to perform oscillation extraction for the set time.

[0043] After the shaking extraction is complete, turn off the constant temperature shaker, open the shaker door, and remove the entire placement plate.

[0044] Finally, the operator aligns the two protrusions on both sides of the wedge-shaped insert with the grooves on the inner walls of the slot and pushes it into the slot. The wedge-shaped surface (i.e., the gradually narrowing slope) of the wedge-shaped insert first contacts the wedge-shaped notch at the bottom of the iron block. As the wedge-shaped insert continues to go deeper, the wedge-shaped surface and the wedge-shaped notch slide relative to each other, applying an upward pushing force to the iron block. This pushing force is used to overcome the magnetic attraction between the iron block and the magnet. Once the magnetic attraction is completely overcome, the iron block separates from the magnet.

[0045] At this point, the return spring is no longer subjected to downward pressure, and its stored elastic potential energy is released, pushing the fixed column upward along the circular groove, and the base moves upward synchronously. After the base moves upward, its lower surface no longer applies pressure to the second clamping parts, and each of the second clamping parts loses its downward constraint. The first clamping part rotates outward, releasing its contact with the outer wall of the conical flask, and the conical flask can be easily removed.

[0046] Example 2

[0047] (1) Chromatographic conditions

[0048] Chromatographic column: NanoChrom C18 (4.6×250mm, 5μm); mobile phase: acetonitrile (phase A) - 0.05% phosphoric acid aqueous solution (phase B); detection wavelength: 283nm and 330nm; column temperature: 30℃; flow rate: 1.0mL / min; injection volume: 10μL.

[0049] The elution procedure is shown in Table 2.

[0050] Table 2 Elution Procedure

[0051] (2) Preparation of mixed reference solution

[0052] Accurately weigh the reference standards of naringin, hesperidin, novo tangeretin and citrus peel according to Table 3, dissolve and dilute them with methanol to obtain the single standard stock solution of the reference standard.

[0053] Table 3 Preparation of Standard Stock Solutions

[0054] According to Table 4, accurately pipette the single-standard stock solutions of each reference standard in sequence, place them in the same volumetric flask, add methanol to the mark, and shake well to obtain a mixed reference standard solution with a relative concentration of 200%. Using the 200% relative concentration mixed reference standard as the stock solution, dilute with 70% methanol to obtain mixed reference standard solutions with relative concentrations of 10% to 200% as shown in Table 5, which are used as mixed reference standard solutions for linearity studies.

[0055] Table 4. Preparation of 200% Relative Concentration Control Solution

[0056] Table 5. Preparation of Linear Mixed Standard Solutions

[0057] According to Table 6, take the corresponding volume of the reference solution from the single standard stock solution (Table 3) and place it in a 50 mL volumetric flask. Add a certain amount of methanol to dissolve it and then dilute to the mark to obtain the mixed reference solution for the spiked recovery experiment.

[0058] Table 6. Preparation of Mixed Standard Solution for Spiked Recovery Experiment

[0059] Following the preparation method in Table 7, take 2 mL of the solution from the spiked recovery test mixed reference solution shown in Table 6 and place it in a 5 mL volumetric flask. Dilute to the mark with methanol to obtain the mixed reference solution used for methodological investigation and sample determination.

[0060] Table 7. Preparation of Mixed Reference Solution for Methodological Investigation and Sample Determination

[0061] (3) Preparation of the test solution

[0062] Accurately weigh 0.2 g of sample powder (passed through a No. 4 sieve), place it in a stoppered conical flask, accurately add 25 mL of pure methanol, seal tightly, weigh, sonicate for 45 minutes, cool to room temperature, weigh again using an extraction device, replenish the lost weight with pure methanol, shake well, let stand, take the supernatant, filter through a 0.45 μm filter membrane, take 10 μ L of the filtrate, mix with 10 μ L of the reference solution, inject into the liquid chromatograph, determine, and calculate.

[0063] (4) Methodological verification

[0064] Determination and verification of relative correction factors

[0065] Accurately pipette the mixed reference solution and serially dilute it with methanol to prepare a series of mixed reference solutions with relative concentrations of 10%, 20%, 40%, 80%, 100%, 160%, and 200%, respectively. Linear mixed reference solutions were also prepared. Inject and determine the peak areas under the chromatographic conditions described above. Plot a standard curve with the mass concentration of each reference standard as the abscissa (x) and the peak area as the ordinate (y), and calculate the regression equation. The results are shown in Table 8.

[0066] Table 8. Linear regression equations for the four components

[0067] The results showed that naringin, hesperidin, novohesperidin and citrus peel had good linear relationships within their respective concentration ranges.

[0068] Using the high-performance liquid chromatograms of the aforementioned series of mixed reference solutions at various concentrations, the peak areas were calculated. With hesperidin as an internal reference, the peak areas of naringin, nobiletin, and citrusin were calculated according to the following formulas. value:

[0069] In the formula, A i C represents the peak area of ​​a certain analyte i; i Let A be the concentration of the analyte i. s C is the peak area of ​​the internal reference s. s The concentration of the internal reference substance s.

[0070] Table 9. Relative correction factors using hesperidin as an internal reference.

[0071] Precision test

[0072] Take 10 μL of the mixed reference solution prepared in Table 7 for methodological investigation, inject it 6 times consecutively under the aforementioned chromatographic conditions, record the peak area, and calculate the RSD value.

[0073] After recording the peak areas, the RSD% of naringin, hesperidin, nobiletin, and citrusin were calculated to be 0.43%, 0.05%, 0.12%, and 0.03%, respectively. This indicates that the method has good precision.

[0074] Repeatability test

[0075] Accurately weigh 0.18g, 0.20g, and 0.22g of cp-11 sample powder (3 portions each), prepare test solutions according to the test sample preparation method, inject and analyze according to the aforementioned chromatographic conditions, record the peak area of ​​the chromatogram, and calculate the RSD value of the peak area of ​​the 9 test samples.

[0076] After recording the peak areas, the contents of naringin, hesperidin, nobiletin, and citrusin in the sample were calculated to be 0.12%, 5.41%, 0.65%, and 0.29%, respectively; their RSD% were 3.28%, 4.47%, 1.76%, and 1.75%, respectively. This indicates that the method has good reproducibility and is feasible.

[0077] Intermediate precision experiment

[0078] Accurately weigh 0.2g of cp-11 sample powder into two portions, prepare the test solution according to the test sample preparation method, and then inject and analyze the sample under the aforementioned chromatographic conditions, and record the chromatogram.

[0079] After recording the peak areas, experimenter A calculated the contents of naringin, hesperidin, nobiletin, and citrus peel in the sample to be 0.12%, 5.45%, 0.65%, and 0.29%, respectively; experimenter B calculated the contents of naringin, hesperidin, nobiletin, and citrus peel in the sample to be 0.12%, 5.63%, 0.64%, and 0.28%, respectively. The relative average deviations between the two sets of data were 1.92%, 3.24%, 1.17%, and 1.25%, respectively.

[0080] Stability test

[0081] Accurately weigh 0.20 g of cp-11 sample powder, prepare the test solution according to the test sample preparation method, and analyze it at 0, 3, 6, 12, 24 and 36 h according to the aforementioned chromatographic conditions, and record the chromatogram.

[0082] After recording the peak areas, the RSD% of the peak areas of naringin, hesperidin, nobiletin, and citrusin at six time points were calculated to be 0.41%, 1.16%, 0.34%, and 0.37%, respectively. The results indicate that the sample maintains good stability within 0–36 hours.

[0083] Spiking recovery experiment

[0084] Nine portions of 0.1 g of cp-11 sample powder were accurately weighed and divided into three batches: low, medium, and high. The mixed reference solution was added at levels of 40%, 50%, and 60%, respectively. The test solution was prepared according to the test sample preparation method and then injected for analysis under the aforementioned chromatographic conditions. The spiked recovery rate was calculated using the mixed reference solution (Table 6) as a control and the external standard method.

[0085] After recording the peak area, the recoveries, mean recoveries, and RSD% of naringin, hesperidin, nosocomioterpenoid, and citrusin in the three batches of samples (low, medium, and high) were calculated and are shown in Table 10.

[0086] Table 10 Results of Spike Recovery Test

[0087] Durability evaluation of relative correction factors

[0088] a. The effects of different chromatograms and different chromatographic columns Impact

[0089] Accurately pipette 1 mL of the mixed reference solution prepared in Table 7, and investigate the components of NanoChrom C18, ShimNex CSC18, and Midspect Red RD-C18 during operation on a Shimadzu SPD-M20A high-performance liquid chromatograph and an Agilent 1200 (DAD) high-performance liquid chromatograph. The changes are shown in Table 11.

[0090] Table 11. Effects of different chromatograms and columns on the performance of different chromatograms The influence of value

[0091] b. Effects of different volumetric flow rates and different column temperatures on Impact

[0092] Accurately pipette the mixed reference solution from Table 7 and use a NanoChrom C18 (4.6×250mm, 5μm) column and a Shimadzu SPD-M20A high-performance liquid chromatograph to investigate the components under different flow rates (0.8, 1.0, 1.2 mL / min) and column temperatures (25, 30, 35℃). The changes are shown in Table 12.

[0093] Table 12 Effects of different volumetric flow rates and column temperatures on column temperature The influence of value

[0094] c. Relative retention time of relative chromatographic peaks ( ) calculation

[0095] Using the relative retention time method ( The formula is as follows:

[0096] in the formula Let i be the retention time of a certain analyte i. is the retention time of the internal reference substance s.

[0097] The retention times of hesperidin, naringin, norihesperidin, and tangeretin in the linear mixed reference solution were recorded using HPLC. The relative retention time method was employed, with hesperidin as an internal reference, to calculate the relative retention times of naringin, norihesperidin, and tangeretin with hesperidin. The chromatographic peaks were located, and the performance of NanoChrom C18, ShimNex CS C18, and Spectrum Red RD-C18 HPLC systems, as well as Shimadzu and Agilent HPLC systems, was investigated. The effects are shown in Table 13.

[0098] Table 13 Effects of different chromatograms and columns on relative retention time

[0099] (5) Sample testing

[0100] Moisture determination

[0101] The toluene method was used to detect the moisture content of the sample. Approximately 25g of sample powder was accurately weighed and placed in a round-bottom flask. 200mL of toluene was added, along with glass beads to prevent bumping. The mixture was heated to detect the moisture content. The following formula was used: Moisture content in sample (%) = (V1 - V0) × ρ / m × 100% Calculate the moisture content in the tangerine peel sample, where V1 is the volume of moisture in the receiving tube, V0 is the volume of moisture in the blank test, ρ is the water density (20℃, ρ=1.00g / mL), and m is the sample mass (g).

[0102] The collected 16 batches of citrus samples were used to prepare test solutions according to the aforementioned method. The samples were injected for testing, and the concentrations (mg / mL) of the four active ingredients in the test solutions and the contents (mg / g) in the medicinal powder were calculated based on the regression equation. The results are shown in Table 14.

[0103] Table 14 Results of effective component content in 16 batches of citrus samples

[0104] Based on the sample test results and chromatograms, hesperidin was used as the internal reference for the QAMS method, and the average relative correction factor corresponding to each component was determined experimentally. Refer to the QAMS quantitative calculation formula:

[0105] In the formula The peak area of ​​the component to be measured. The content of the component to be measured. The peak area of ​​the internal reference material. This refers to the content of internal reference substances. This is the relative correction factor between the analyte and the internal reference.

[0106] The contents of the other three components in the sample were calculated separately, and the results of QAMS and ESM determinations were compared, as shown in Table 15.

[0107] Table 15. Content determination results using the external standard method and the one-test-multiple-evaluation method.

[0108] The results showed that the relative deviations of different batches of samples for each component were all within a reasonable range, with no obvious systematic deviations. This indicates that the results of the one-test-multiple-evaluation method and the external standard method were similar, and the two quantitative methods had good consistency in accuracy. The established one-test-multiple-evaluation method can be used for simultaneous quality evaluation of multiple components in tangerine peel.

[0109] Comparative Example 1

[0110] In this comparative example, unlike Example 1, the extraction device was omitted, and only manual liquid replenishment and weighing were performed.

[0111] Methodological investigation revealed that, in stability tests, the two replenishment methods performed consistently with no significant differences in the tests examining only the chemical stability of the solution. However, in the spiked recovery data, the RSD% for naringin, hesperidin, nobiletin, and citrusin were 2.52%, 2.41%, 3.23%, and 2.83%, respectively. This indicates that the RSD of the recovery rate was generally higher with manual replenishment; the RSD <2% in Example 2 reflects the high level of precision achieved through mechanical replenishment.

[0112] As can be seen from the above embodiments, the present invention provides a quantitative analysis method for multiple components in dried tangerine peel using a single assay and multiple evaluation method. Example 2 shows that the present invention exhibits good linearity, precision, repeatability, stability, and recovery rate. The results of the single assay and multiple evaluation method are not significantly different from those of the external standard method (relative deviation < 2.5%), indicating that the method is accurate and reliable. In Comparative Example 1, the RSD of the recovery rate of the manual replenishment group increased to over 2.5%, while the RSD of the automatic replenishment device group of the present invention was < 2%, proving that the device significantly reduces human error and improves precision.

[0113] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A quantitative analysis method for multiple components in dried tangerine peel, characterized in that, Includes the following steps: 1) Accurately weigh the naringin standard, hesperidin standard, norihesperidin standard and citrus peel standard, mix them with solvent and dilute them to obtain a series of mixed reference solutions of different concentrations; 2) Accurately weigh the tangerine peel powder and place it in an Erlenmeyer flask. Accurately add the extractant, and after ultrasonic extraction, replenishment, shaking and filtration, the test solution is obtained. 3) Inject the series of mixed reference solutions and test solutions separately under chromatographic conditions and record the peak area of ​​each component; Plot a standard curve with the mass concentration of each reference standard as the x-axis and the peak area as the y-axis, and calculate the regression equation. 4) The method of one test and multiple evaluations was adopted, and hesperidin was used as an internal reference. The content of each analyte was calculated according to the formula of relative correction factor. In step 2), the replenishment and mixing are performed in an extraction device, which includes: The platform is equipped with a constant temperature shaking table; A clamp is disposed inside the constant temperature shaker and is used to fix the conical flask; An electronic scale is installed on the platform and located on one side of the constant temperature shaker. A tray is fixedly installed on the electronic scale for placing conical flasks to be replenished. A solvent replenishment mechanism, mounted on the platform, is used to replenish the solvent into the conical flask to make up for the lost weight.

2. The quantitative analysis method for multiple components in dried tangerine peel using a single measurement and multiple evaluation method according to claim 1, characterized in that, In step 1), the solvent includes methanol or an aqueous methanol solution; In the series of mixed reference solutions, the concentrations of naringin, hesperidin, nobiletin, and citrusin were 3-65 μg / mL, 25-510 μg / mL, 3-65 μg / mL, and 1-35 μg / mL, respectively.

3. A quantitative analysis method for multiple components in dried tangerine peel using a single measurement and multiple evaluation method according to claim 1 or 2, characterized in that, In step 2), the extractant includes methanol or an aqueous methanol solution; The ratio of tangerine peel powder to extractant is 0.1~0.4g:25~30mL.

4. The quantitative analysis method for multiple components in dried tangerine peel using a single measurement and multiple evaluation method according to claim 3, characterized in that, In step 3), the chromatographic conditions are as follows: Chromatographic column: C18 column; Mobile phase: acetonitrile-0.05% phosphoric acid solution; Detection wavelength: 280~330nm; Column temperature: 25~30℃; Flow rate: 0.8~1.2 mL / min; Gradient elution program: 0-15 min, 10% acetonitrile; 15-16 min, 35% acetonitrile; 16-40 min, 40% acetonitrile; 40-46 min, 60% acetonitrile; 46-50 min, 10% acetonitrile.

5. The quantitative analysis method for multiple components in dried tangerine peel using a single measurement and multiple evaluation method according to claim 1, characterized in that, In step 4), the formula for calculating the relative correction factor is as follows: ， Among them, A i C represents the peak area of ​​a certain analyte i; i Let A be the concentration of the analyte i. s C is the peak area of ​​the internal reference s. s The concentration of the internal reference substance s.

6. The quantitative analysis method for multiple components in dried tangerine peel using a single measurement and multiple evaluation method according to claim 1, characterized in that, In the extraction device, the clamp includes a placement plate and a plurality of clamp bodies arranged in an array on the placement plate. The placement plate has a set of grooves, each of which includes a circular groove and a plurality of elongated grooves arranged circumferentially around the circular groove. The side of the placement plate has a slot, which is located below the circular groove and communicates with the circular groove. The inner walls on both sides of the slot have sliding grooves, and a magnet is fixed inside the slot.

7. The quantitative analysis method for multiple components in dried tangerine peel using a single measurement and multiple evaluation method according to claim 6, characterized in that, The clamp body includes: Multiple clamping plates are provided, each corresponding to one of the elongated grooves. Each clamping plate is an elastically bent component, comprising a first clamping part and a second clamping part connected at an acute angle, and the connection between the two parts is rotatably mounted on one side edge of the corresponding elongated groove. The first clamping part is used to abut against the outer wall of the conical bottle, and the second clamping part is used to abut against the lower surface of the placement plate in the initial state. The base has an outer contour that matches the circular groove, and the base abuts against the upper surface of each of the second clamping parts; a fixing post is fixedly connected to the center bottom of the base, the fixing post passes through the circular groove and extends into the corresponding slot, and an iron block is fixed to the bottom end of the fixing post, and a wedge-shaped notch is opened at the bottom end of the iron block; A return spring is sleeved on the outer periphery of the fixed column and elastically supported between the bottom wall of the circular groove and the lower surface of the base. A wedge-shaped insert plate, wherein both sides of the wedge-shaped insert plate are provided with protrusions that slide in conjunction with the groove; When the wedge-shaped insert is pushed into the slot, the wedge-shaped surface of the wedge-shaped insert abuts against the wedge-shaped notch to push the iron block to overcome the attraction of the magnet and separate. At the same time, the return spring releases the elastic force and pushes the fixing column to move upward, so that the base moves upward and each of the clamps rotates outward around its connection to release the conical bottle. Each of the first clamping parts is equipped with a rubber pad.

8. The quantitative analysis method for multiple components in dried tangerine peel using a single measurement and multiple evaluation method according to claim 1, characterized in that, The solvent replenishment mechanism includes: A support frame, wherein a liquid outlet is provided on the support frame and the liquid outlet is located directly above the tray; A solvent storage tank is disposed on the side of the bracket away from the electronic scale; A pump is provided on one side of the solvent storage tank, with its inlet end connected to the solvent storage tank and its outlet end connected to the outlet via a pipe. A metering valve, which is installed on the pipeline, is used to control the amount of solvent replenished.

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