Fingerprint spectrum of citrus chachiensis hortorum and construction method and application thereof
The fingerprint spectrum of Guangchenpi was constructed by headspace-gas chromatography-mass spectrometry, which solved the problem of identifying the origin and year of Guangchenpi and enabled rapid and accurate quality control and traceability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-27
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies make it difficult to quickly, comprehensively, and accurately identify the origin and year of Guangchenpi (a type of dried tangerine peel), resulting in problems such as mixed varieties, counterfeit origins, and falsely labeled years in the market.
The volatile components of Guangchenpi were extracted and analyzed by headspace gas chromatography-mass spectrometry, a fingerprint spectrum of Guangchenpi was constructed, and the mass spectra of characteristic compounds were extracted by unknown substance analysis software to establish a quality control method for Guangchenpi.
It enables accurate and comprehensive quality control of Guangchenpi (dried tangerine peel), provides scientific quality evaluation and traceability basis, and can quickly identify and distinguish the place of origin and year of Guangchenpi.
Smart Images
Figure CN121762744A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical testing technology, specifically relating to a fingerprint spectrum of dried tangerine peel, its construction method, and its application. Background Technology
[0002] Guangchenpi, specifically referring to dried tangerine peel produced in Guangdong, China, with Xinhui as its core production area, is made from the dried, mature peel of the Rutaceae citrus fruit, which is then aged. It is not only a historically significant medicinal and edible product but also the foremost of the "Three Treasures of Guangdong," with "the older the better" as its core value. In traditional Chinese medicine theory, Guangchenpi has the effects of regulating qi and strengthening the spleen, as well as drying dampness and resolving phlegm. Its quality improves with aging, as the volatile oils and flavonoids (such as hesperidin and norihesperidin) continuously transform and synergize, forming a unique, mellow aroma and significant pharmacological effects.
[0003] However, with surging market demand and rising brand value, the Guangchenpi market faces prominent problems such as mixed varieties, misrepresentation of origin, and false labeling of vintage. To address this chaos, the industry and academia have developed and applied various identification methods, but all have certain limitations.
[0004] Firstly, traditional experience-based identification methods rely primarily on the sensory experience of practitioners. They determine the origin and approximate year by observing the appearance (skin shape, oil cells), color, aroma, taste, and texture. This method is simple and intuitive, serving as a basic tool in the distribution process. However, it is highly dependent on subjective experience, difficult to quantify, and susceptible to human error, making it unable to accurately identify counterfeit products or scientifically define specific years. While physicochemical testing, such as determining hesperidin content (a legal standard in the Chinese Pharmacopoeia), ensures basic medicinal quality, a single indicator cannot distinguish specific origins or aging years, making it difficult to objectively evaluate the intrinsic quality of Guangchenpi (Guangdong aged tangerine peel).
[0005] Therefore, developing a rapid, comprehensive, and accurate quality control method has become an urgent need to regulate the Guangchenpi market and ensure its quality and reputation. Summary of the Invention
[0006] The purpose of this invention is to provide a fingerprint spectrum of Guangchenpi (dried tangerine peel), its construction method, and its application. The fingerprint spectrum of Guangchenpi in this invention uses multiple volatile substances as quality indicators of Guangchenpi, which can achieve accurate and comprehensive quality control of Guangchenpi.
[0007] The technical solution adopted to achieve the above objectives is to provide a method for constructing a fingerprint spectrum of dried tangerine peel, including the following steps: The volatile components of Guangchenpi were extracted and analyzed by headspace-gas chromatography-mass spectrometry. The mass spectra of characteristic compounds were extracted by unknown substance analysis software, thus obtaining the fingerprint spectrum of Guangchenpi. The detection conditions for headspace-gas chromatography-mass spectrometry include: (1) Solid headspace is used. Take dried tangerine peel and seal it in the headspace bottle. The equilibrium temperature is 80~90℃, the injection needle temperature is 85~95℃, the equilibrium time is 35~45 min, the quantitative loop pressure is stabilized in the default mode, the injection time is 1.5~2.5 min, and the GC cycle time is 45~50 min. (2) Gas chromatography uses a capillary column with helium as the carrier gas and a column flow rate of 1.0~1.2 mL / min; the injection port temperature is 255~265℃, split injection is used, the injection volume is 0.2~0.3 mL, and the split ratio is 2~5:1; the column temperature program is as follows: the initial temperature is 48~52℃, held for 4~6 min, then increased to 70℃ at 0.8~1.2℃ / min, then increased to 155~165℃ at 4.8~5.2℃ / min, and then increased to 250~270℃ at 10~20℃ / min, and the test ends. (3) The mass spectrometry ionization method is electron bombardment voltage, 65~75 eV, ion source temperature is 220~240℃, quadrupole temperature is 145~155℃, GC-MS interface temperature is 250~270℃, solvent delay time is 1.5~2.5 min, and scanning mode is full scan 35~550 m / z.
[0008] Preferably, Guangchenpi includes green peel, second-ripe peel and big-ripe peel at different maturity stages, with a total of ≥45 pieces.
[0009] More preferably, Guangchenpi includes fresh fruit peel and dried peels at different maturity stages, namely green peel, second red peel and big red peel, with a total of 52 types.
[0010] Preferably, step (1) includes the following steps: using solid headspace, taking dried tangerine peel into a headspace bottle and sealing it, equilibration temperature 90℃, injection needle temperature 95℃, equilibration time 45 min, quantitative loop pressure stabilization using default mode, injection time 2 min, GC cycle time 48 min.
[0011] More preferably, in step (1), the size of the dried tangerine peel is 0.5×2 cm and the weight is 0.05~0.2 g.
[0012] Preferably, in step (2), the column flow rate is 1.0 mL / min; the injection port temperature is 260℃, split injection is used, the injection volume is 0.25 mL, and the split ratio is 5:1; the column temperature program is as follows: initial temperature 50℃, hold for 5 min, then increase to 70℃ at 1℃ / min, then increase to 160℃ at 5℃ / min, and then increase to 260℃ at 15℃ / min, and the test ends.
[0013] More preferably, the capillary column is model BD-5ms with dimensions of 30 m × 0.25 mm × 0.25 µm.
[0014] Preferably, step (3) includes the following steps: the mass spectrometry ionization method is electron bombardment voltage, 70 eV, the ion source temperature is 230℃, the quadrupole temperature is 150℃, the GC-MS interface temperature is 260℃, the solvent delay time is 2 min, and the scanning mode is full scan 35~550 m / z.
[0015] More preferably, the unknown substance analysis software is Agilent MassHunter unknown substance analysis software, which uses deconvolution to extract the mass spectrum.
[0016] This invention also provides a fingerprint spectrum of Guangchenpi (dried tangerine peel) constructed using a Guangchenpi fingerprint spectrum construction method. The Guangchenpi fingerprint spectrum consists of 42 common fingerprint peaks, and the relative retention times of the 42 common fingerprint peaks in the fingerprint spectrum are: .
[0017] This invention also provides the application of Guangchenpi fingerprint spectrum in the identification and traceability of Guangchenpi.
[0018] The present invention has the following beneficial effects: This invention utilizes HS-GC-MS technology to analyze and identify common volatile components in Guangchenpi samples. Based on this, a standard fingerprint spectrum of volatile components in Guangchenpi and a mass spectrometry library of corresponding compounds were established. This spectral library constitutes the basic database for the identification and traceability of Guangchenpi, and can provide a scientific basis for the quality evaluation and control of Guangchenpi. Attached Figure Description
[0019] Figure 1 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 01; (b) is sample 02; (c) is sample 03; (d) is sample 04; and (e) is sample 05. Figure 2 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 06; (b) is sample 07; (c) is sample 08; (d) is sample 09; and (e) is sample 10. Figure 3 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 11; (b) is sample 12; (c) is sample 13; (d) is sample 14; and (e) is sample 15. Figure 4The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 16; (b) is sample 17; (c) is sample 18; (d) is sample 19; and (e) is sample 20. Figure 5 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 21; (b) is sample 22; (c) is sample 23; (d) is sample 24; and (e) is sample 25. Figure 6 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 26; (b) is sample 27; (c) is sample 28; (d) is sample 29; and (e) is sample 30. Figure 7 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 31; (b) is sample 32; (c) is sample 33; (d) is sample 34; and (e) is sample 35. Figure 8 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 36; (b) is sample 37; (c) is sample 38; (d) is sample 39; and (e) is sample 40. Figure 9 Here are the GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples; where (a) is sample 41; (b) is sample 42; (c) is sample 43; (d) is sample 44; and (e) is sample 45. Figure 10 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown below; (a) is sample 46; (b) is sample 47; (c) is sample 48; (d) is sample 49; and (e) is sample 50. Figure 11 The GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples are shown; (a) is sample 51; (b) is sample 52. Figure 12 For the fingerprint spectrum of dried tangerine peel; Figure 13 GC-MS characteristic chromatograms of volatile compounds in Guangchenpi samples under different injection split ratios; where (a) is splitless; (b) is split ratio 2:1; (c) is split ratio 5:1; (d) is split ratio 10:1; and (e) is split ratio 20:1. Figure 14 This is an image of the market-purchased sample from Example 4; Figure 15The following are GC-MS characteristic chromatograms of volatile compounds from commercially purchased samples in Example 4; where (a) is sample S-1; (b) is sample S-2; (c) is sample S-3; (d) is sample S-4; and (e) is sample S-5. Figure 16 The GC-MS characteristic chromatograms of the volatile compounds in the commercially purchased samples in Example 4 are shown below; (a) is sample S-6; (b) is sample S-7; (c) is sample S-8; and (d) is sample S-9. Figure 17 The GC-MS characteristic chromatograms of the volatile compounds in the commercially purchased samples in Example 4 are shown below; (a) is sample S-10-1; (b) is sample S-10-2; (c) is sample S-10-3; and (d) is sample S-10-4. Figure 18 The GC-MS characteristic chromatograms of the volatile compounds in the commercially purchased samples in Example 4 are shown below; (a) is sample S-11-1; (b) is sample S-11-2; (c) is sample S-11-3; and (d) is sample S-11-4. Figure 19 The GC-MS characteristic chromatograms of the volatile compounds in the commercially purchased samples in Example 4 are shown below; (a) is sample S-12; and (b) is sample S-13. Detailed Implementation
[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.
[0021] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0022] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0023] Example 1 1. Samples and reagents The instruments used in this invention are: a 7890B-5977 gas chromatograph-quadrupole mass spectrometer (Agilent Technologies, Inc.), equipped with a CTC injection device and an EI source; 20 mL headspace vials; and a part per ten thousand electronic balance.
[0024] The samples were made from fresh fruit peels and dried tangerine peels sold in the market. Detailed information is shown in Table 1 below.
[0025] Table 1 Sample Information Table
[0026] 2. Test Methods 2.1 Determination of volatile components in the sample by extraction The sample of Guangchenpi included green tangerine peel, second red tangerine peel, and big red tangerine peel, three different types of tangerine peel at different maturity stages. A whole piece of the above sample tangerine peel, 0.05~0.2 g, measuring 0.5×2 cm, was placed in a headspace vial, and the cap was immediately tightened. The volatile substances of the sample were extracted under constant temperature equilibrium and then tested by headspace injection.
[0027] 2.2 Headspace Sampler Conditions Sample equilibrium temperature 90℃, injection needle temperature 95℃, sample equilibrium time 45 min, quantitative loop pressure stabilization in default mode, injection time 2 min, GC cycle time 48 min.
[0028] 2.3 Gas Chromatography Determination Conditions Chromatographic column: BD-5ms capillary column (30 m × 0.25 mm × 0.25 µm), carrier gas is high-purity helium, column flow rate is constant 1.0 mL / min; injection port temperature is 260℃, split injection is used, injection volume is 0.25 mL, split ratio is 5:1; column temperature program is as follows: initial temperature 50℃, hold for 5 min, then increase to 70℃ at 1℃ / min, then increase to 160℃ at 5℃ / min, then increase to 260℃ at 15℃ / min, and the test ends.
[0029] 2.4 Mass Spectrometry Conditions The ionization method was electron impact voltage of 70 eV, the ion source temperature was 230℃, the quadrupole temperature was 150℃, the GC-MS interface temperature was 260℃, the solvent delay time was 2 min, and the scanning mode was full scan 35-550 m / z.
[0030] 3. Analysis of Spectral Results Gas chromatography-mass spectra are shown below. Figures 1-11 Analysis was performed using Agilent MassHunter software. The chromatograms of each compound were deconvolved to extract the corresponding mass spectra, which were then compared with the NIST spectral library for compound identification. Under the same display conditions, the sample chromatograms were opened and superimposed to obtain the GC-MS characteristic chromatograms of the volatile components of dried tangerine peel. (See attached image.) Figure 12A total of 42 chromatographic peaks were observed, with highly similar mass spectrometric peaks. Under the chromatographic conditions used, compounds with higher content were observed in the first 25 minutes, while those with lower content were observed after 25 minutes. The relative content of volatile components varied among compounds from different origins, and there were also some differences among compounds observed after 25 minutes.
[0031] 4. Identification of volatile compounds in dried tangerine peel By comparing the characteristic total ion chromatograms of volatile components in the samples using GC-MS ( Figure 12 The mass spectra of each peak were compared with the standard mass spectra of NIST 2023 and standard substances, and qualitative analysis was performed in conjunction with the mass spectra and literature data. Forty-two distinct common color peaks were observed. The first 25 min contained the main components of the volatile oil from Guangchenpi (Citrus reticulata peel), accounting for 98% of the total content, including eight compounds, mainly alkenes, with D-limonene being the most abundant, followed by γ-terpinene. The compounds present after 25 min were less abundant but more diverse. Methyl 2-(methylamino)benzoate, with a retention time of 38.7 min, is a unique chemical component of Guangchenpi (Citrus reticulata peel). The identified compounds are shown in Table 2.
[0032] Table 2 Analysis of volatile compounds
[0033] Example 2: Selection of Equilibrium Temperature for Extraction of Volatile Substances from Samples To understand the types and contents of volatile components in dried tangerine peel, the extraction temperature and time were investigated. The headspace sampler conditions, including sample equilibrium temperature and time, were adjusted to 80, 85, 90, and 95°C, and extraction times of 30, 45, 50, and 60 min. The peak area per unit mass of seven compounds—α-pinene, β-pinene, β-myrcene, p-isophenyltoluene, D-limonene, γ-terpinene, and methyl 2-(methylamino)benzoate—was measured. Other parameters and conditions were the same as in Example 1. The results are shown in Tables 3 and 4.
[0034] Table 3. Changes in seven volatile components at equilibrium temperature in different samples.
[0035] Table 4. Changes in seven volatile components at equilibrium time in different samples.
[0036] Testing revealed that seven compounds—α-pinene, β-pinene, β-myrcene, p-isophenyltoluene, D-limonene, γ-terpinene, and methyl 2-(methylamino)benzoate—achieved maximum extraction efficiency at 80℃. However, peak elution occurred after 25 min. Compounds with lower content, such as methyl 2-methylaminobenzoate, showed the largest peak area at 90℃. Considering all factors, the optimal equilibrium temperature was determined to be 90℃, with a sample equilibrium time of 45 min.
[0037] Example 3 Selection of Gas Chromatography Determination Conditions The volatile components of dried tangerine peel are mainly alkenes, with α-pinene, β-pinene, β-myrcene, p-isophenyltoluene, D-limonene, γ-terpinene, and methyl 2-methylaminobenzoate being the main components, accounting for over 98% of all volatile components. The experiment involved testing and adjusting different injection splits using gas chromatography; other parameters and conditions were the same as in Example 1. The obtained chromatogram is shown below. Figure 13 .
[0038] from Figure 13 It can be seen that if the injection volume is too small during testing, compounds with low content, such as methyl 2-(methylamino)benzoate, cannot be detected. When the injection volume is large, sample overload is more likely to occur, and the chromatographic peak will be distorted. By comparing the effects of different injection split ratios on the peaks of each compound, when the injection split ratio is greater than 10:1, some compounds that elute after 24 min are basically undetectable due to their low content. Considering the resolution and peak shape of each compound, a split injection with a split ratio of 5:1 is adopted.
[0039] Example 4: Application of Guangchenpi fingerprint spectrum in the identification and traceability of Guangchenpi In practical applications, the HS-GC-MS detection results of the sample to be tested are imported into Agilent MassHunter quantitative similarity software and compared with the standard fingerprint spectrum obtained in the examples. The similarity value is calculated for determination. The higher the similarity, the stronger the consistency between the sample and the standard Guangchenpi (aged tangerine peel), thereby achieving rapid identification and preliminary traceability.
[0040] Thirteen samples were purchased from the market, totaling 19 items, numbered S-1 to S-13. The actual appearance of the samples can be seen in [the image / document / etc.]. Figure 14 The volatile components were detected using the method described in Example 1. Similarity analysis was performed using the established fingerprint spectrum of volatile components in Guangchenpi (dried tangerine peel). The results are as follows: Figures 15-19 .
[0041] The results showed that the Guangxi Zhuang Autonomous Region's Dahongpi and Qingpi varieties, along with Xinhui's Guangchenpi, are all made from dried tea branch tangerine peel. Their volatile components contain methyl 2-(methylamino)benzoate. The compounds in the first 25 minutes are the same as those in Xinhui Guangchenpi, and the content ratios are also similar. However, the total oil content is lower, and the trace compounds after 25 minutes are less abundant. This could be used to differentiate the material basis between the two different origins. Sample S-9 was claimed to be 5-year-old Guangchenpi, but its volatile component chromatogram differed significantly from the fingerprint of Xinhui Guangchenpi, and methyl 2-(methylamino)benzoate was not present. Its chromatogram was similar to the chromatograms of four components from ordinary tangerine peel S-10, suggesting that this sample may be dried ordinary tangerine peel, not Guangchenpi made from dried tea branch tangerine peel.
[0042] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.
Claims
1. A method for constructing a fingerprint spectrum of dried tangerine peel, characterized in that, Includes the following steps: The volatile components of Guangchenpi were extracted and analyzed by headspace-gas chromatography-mass spectrometry. The mass spectra of characteristic compounds were extracted by unknown substance analysis software, thus obtaining the fingerprint spectrum of Guangchenpi. The headspace-gas chromatography-mass spectrometry (HCGC-MS) detection conditions include: (1) Solid headspace is used. Take dried tangerine peel and seal it in the headspace bottle. The equilibrium temperature is 80~90℃, the injection needle temperature is 85~95℃, the equilibrium time is 35~45 min, the quantitative loop pressure is stabilized in the default mode, the injection time is 1.5~2.5 min, and the GC cycle time is 45~50 min. (2) Gas chromatography uses a capillary column with helium as the carrier gas and a column flow rate of 1.0~1.2 mL / min; the injection port temperature is 255~265℃, split injection is used, the injection volume is 0.2~0.3 mL, and the split ratio is 2~5:1; the column temperature program is as follows: the initial temperature is 48~52℃, held for 4~6 min, then increased to 70℃ at 0.8~1.2℃ / min, then increased to 155~165℃ at 4.8~5.2℃ / min, and then increased to 250~270℃ at 10~20℃ / min, and the test ends. (3) The mass spectrometry ionization method is electron bombardment voltage, 65~75 eV, ion source temperature is 220~240℃, quadrupole temperature is 145~155℃, GC-MS interface temperature is 250~270℃, solvent delay time is 1.5~2.5 min, and scanning mode is full scan 35~550 m / z.
2. The method for constructing a fingerprint spectrum of dried tangerine peel as described in claim 1, characterized in that, The Guangchenpi includes green peel, second-ripe peel and large-ripe peel at different maturity stages, with a total of ≥45 pieces.
3. The method for constructing a fingerprint spectrum of dried tangerine peel as described in claim 1, characterized in that, Step (1) includes the following steps: using solid headspace, taking dried tangerine peel into the headspace bottle and sealing it, equilibration temperature 90℃, injection needle temperature 95℃, equilibration time 45 min, quantitative loop pressure stabilization using the default mode, injection time 2 min, GC cycle time 48 min.
4. The method for constructing a fingerprint spectrum of dried tangerine peel as described in claim 3, characterized in that, In step (1), the size of the dried tangerine peel is 0.5×2 cm and the weight is 0.05~0.2 g.
5. The method for constructing a fingerprint spectrum of dried tangerine peel as described in claim 1, characterized in that, In step (2), the column flow rate is 1.0 mL / min; the injection port temperature is 260℃, split injection is used, the injection volume is 0.25 mL, and the split ratio is 5:1; the column temperature program is as follows: initial temperature 50℃, hold for 5 min, then increase to 70℃ at 1℃ / min, then increase to 160℃ at 5℃ / min, and then increase to 260℃ at 15℃ / min, and the test ends.
6. The method for constructing a fingerprint spectrum of dried tangerine peel as described in claim 1 or 5, characterized in that, The capillary column is model BD-5ms, with dimensions of 30 m × 0.25 mm × 0.25 µm.
7. The method for constructing a fingerprint spectrum of dried tangerine peel as described in claim 1, characterized in that, Step (3) includes the following steps: the mass spectrometry ionization method is electron bombardment voltage, 70 eV, the ion source temperature is 230℃, the quadrupole temperature is 150℃, the GC-MS interface temperature is 260℃, the solvent delay time is 2 min, and the scanning mode is full scan 35~550 m / z.
8. A fingerprint spectrum of Guangchenpi (dried tangerine peel) constructed using the fingerprint spectrum construction method according to any one of claims 1 to 7, characterized in that, The fingerprint spectrum of Guangchenpi (dried tangerine peel) consists of 42 common fingerprint peaks, and the relative retention times of the 42 common fingerprint peaks are as follows: 。 9. The application of the fingerprint spectrum of Guangchenpi as described in claim 8 in the identification and traceability of Guangchenpi.