阅读说明：本技术 一种区分生青皮和醋青皮的质量评价方法 (Quality evaluation method for distinguishing green tangerine peels from vinegar tangerine peels ) 是由 李慧芬 崔伟亮 张学兰 赵盼 刘青芝 于 2021-09-22 设计创作，主要内容包括：本发明属于材料分析领域,具体涉及一种区分生青皮和醋青皮的质量评价方法。该评价方法通过以下步骤实现：制备供试品溶液和对照品溶液,利用高效液相色谱分别建立生青皮和醋青皮的指纹图谱,通过指纹谱图进行生青皮和醋青皮区分验证。本发明系统研究青皮醋炙前后化学成分的变化,筛选醋青皮的质量标志物,为制定更加合理的醋青皮质量标准和进一步研究醋炙青皮的炮制原理提供科学依据；该方法稳定可靠,建立的指纹图谱分析方法能够系统地反映生青皮、醋青皮饮片化学成分的差异,筛选出的新增成分可以作为区分生青皮、醋青皮的质量标志物,为制定更加合理的醋青皮质量标准和进一步研究醋炙青皮的炮制原理提供科学依据。(The invention belongs to the field of material analysis, and particularly relates to a quality evaluation method for distinguishing green tangerine peels from vinegar tangerine peels. The evaluation method is realized by the following steps: preparing a test solution and a reference solution, respectively establishing fingerprint spectrums of green tangerine orange peel and vinegar green tangerine orange peel by using high performance liquid chromatography, and performing differentiation verification on the green tangerine orange peel and the vinegar green tangerine orange peel by using the fingerprint spectrums. The invention systematically studies the change of chemical components before and after vinegar processing of the green tangerine peel, screens the quality marker of the vinegar green tangerine peel, and provides scientific basis for formulating more reasonable quality standard of the vinegar green tangerine peel and further studying the processing principle of the vinegar processed green tangerine peel; the method is stable and reliable, the established fingerprint spectrum analysis method can systematically reflect the difference of chemical components of the green tangerine orange peel and the vinegar green tangerine orange peel decoction pieces, the screened new components can be used as quality markers for distinguishing the green tangerine orange peel and the vinegar green tangerine orange peel, and scientific basis is provided for formulating more reasonable quality standard of the vinegar green tangerine orange peel and further researching the processing principle of the vinegar roasted green tangerine orange peel.)

1. A quality evaluation method for distinguishing green tangerine peels from vinegar tangerine peels is characterized by comprising the following steps:

(1) preparation of a test solution: taking green tangerine peel or vinegar tangerine peel sample powder, precisely weighing, precisely adding methanol, then weighing, ultrasonically treating, cooling, complementing weight loss, filtering, taking subsequent filtrate, and filtering with a microporous filter membrane to obtain the product;

(2) preparation of control solutions: respectively weighing synephrine and hesperidin, precisely weighing, adding methanol to respectively prepare reference substance stock solutions, precisely sucking the reference substance stock solutions, adding methanol to dilute to a constant volume, and preparing into a mixed reference substance solution;

(3) establishing fingerprint spectrums of green tangerine orange peel and vinegar tangerine orange peel respectively by utilizing high performance liquid chromatography;

(4) and performing differentiation verification on green tangerine peels and vinegar tangerine peels through a fingerprint spectrogram.

2. The quality evaluation method according to claim 1, wherein in the step (1), the sample solution is prepared by a specific process comprising: taking about 0.20 g of sample powder, precisely weighing, placing in a 50 mL conical flask with a plug, precisely adding 25mL of 100% methanol, sealing the plug, weighing, performing ultrasonic treatment (100W, 40 KHz) for 30 min, cooling, complementing weight loss, filtering, taking the subsequent filtrate, and filtering with a 0.45 mu m microporous membrane.

3. The quality evaluation method according to claim 1 or 2, wherein in the step (2), the specific preparation process of the reference solution is as follows: respectively weighing appropriate amounts of synephrine and hesperidin, precisely weighing, adding methanol to prepare control stock solutions containing 1.10 mg of synephrine and 0.335 mg of hesperidin per 1 mL respectively, precisely sucking 1 mL of the control stock solutions respectively, placing the control stock solutions in a 25mL volumetric flask, adding methanol to dilute and fix the volume, and preparing a mixed control solution containing 44.00 mu g of synephrine and 13.40 mu g of hesperidin per 1 mL.

4. The quality evaluation method according to claim 1, wherein the conditions of the high performance liquid chromatography are: a chromatographic column: thermo Hypersil GOLD C₁₈Chromatography column (250X 4.6 mm, 5 μm); mobile phase: acetonitrile (a) -0.1% phosphoric acid water (B); sample introduction volume: 10 mu L of the solution; flow rate: 1 mL/min; column temperature: 35 ℃; detection wavelength: 283nm, gradient elution was performed.

5. The quality evaluation method according to claim 4, wherein the conditions of the gradient elution are: 10-14% of A for 0-8 min; 8-17 min, 14% -16% of A; 17-38 min, 16% -18% of A; 38-50 min, 18% -30% A; 30-38% of A for 50-72 min; 72-88 min, 38% -50% A; 88-88.01 min, 50% -10% A; 88.01-98 min, 10% A.

6. A quality evaluation method according to any one of claims 1 to 5, wherein the quality marker for distinguishing green tangerine orange peel from vinegar tangerine orange peel is peak No. 1 and peak No. 5.

Technical Field

The invention belongs to the field of material analysis, and particularly relates to a quality evaluation method for distinguishing green tangerine peels from vinegar tangerine peels.

Background

The pericarpium Citri Reticulatae viride is Citrus reticulata Blanco of RutaceaeCitrus reticulataThe pericarp of dry young fruit or immature fruit of Blanco and its cultivar has effects of dispersing stagnated liver qi, removing qi stagnation, resolving food stagnation, and can be used for treating chest and hypochondrium distending pain, hernia pain,

mammary nodules, mammary abscess, food stagnation and qi stagnation, distending pain in the stomach and abdomen^[1]. Tangerine peel, pericarpium Citri Reticulatae viride, recorded in Song Dynasty, the third cause, the extreme first disease

The syndrome/prescription treatise on "boiling with rice vinegar" contains two decoction pieces of pericarpium Citri Reticulatae viride and pericarpium Citri Reticulatae viride processed with vinegar in the 2020 edition of Chinese pharmacopoeia,

however, the quality standards of the two decoction pieces are different from the content limits of the hesperidin (the hesperidin content in the green tangerine peel is not less than 4.0%, and the hesperidin content in the vinegar green tangerine peel is not less than 3.0%) except for the appearance characteristics and the content limits of the hesperidin, and no clear quality marker of the vinegar green tangerine peel exists, so that the powder of different processed products of the green tangerine peel with the missing appearance characteristics or the Chinese patent medicine containing different processed products of the green tangerine peel can not be effectively distinguished. Modern researches find that pericarpium citri reticulatae viride mainly contains active ingredients such as volatile oil (D-limonene), flavonoid (hesperidin), alkaloid (synephrine) and the like, and the contents of the volatile oil, the hesperidin and the synephrine are reduced after vinegar roasting. King dazzling et al adopts HPLC to determine fingerprint before and after vinegar-roasting of green tangerine peel, identify hesperidin, consider that the difference between fingerprint and relative peak area before and after vinegar-roasting of green tangerine peel is small, and can not effectively distinguish vinegar green tangerine peel from raw green tangerine peel.

Disclosure of Invention

Aiming at the technical problem that the vinegar green tangerine orange peel and raw green tangerine orange peel can not be effectively distinguished in the prior art, the invention provides a quality evaluation method for distinguishing the raw green tangerine orange peel and the vinegar green tangerine orange peel.

The technical scheme adopted by the invention for realizing the purpose is as follows:

the invention provides a quality evaluation method for distinguishing green tangerine peels from vinegar tangerine peels, which comprises the following steps:

(1) preparation of a test solution: taking green tangerine peel or vinegar tangerine peel sample powder, precisely weighing, precisely adding methanol, then weighing, ultrasonically treating, cooling, complementing weight loss, filtering, taking subsequent filtrate, and filtering with a microporous filter membrane to obtain the product;

(2) preparation of control solutions: respectively weighing synephrine and hesperidin, precisely weighing, adding methanol to respectively prepare reference substance stock solutions, precisely sucking the reference substance stock solutions, adding methanol to dilute to a constant volume, and preparing into a mixed reference substance solution;

(3) establishing fingerprint spectrums of green tangerine orange peel and vinegar tangerine orange peel respectively by utilizing high performance liquid chromatography;

(4) and performing differentiation verification on green tangerine peels and vinegar tangerine peels through a fingerprint spectrogram.

The specific preparation process of the test solution provided by the invention comprises the following steps: taking about 0.20 g of sample powder, precisely weighing, placing in a 50 mL conical flask with a plug, precisely adding 25mL of 100% methanol, sealing the plug, weighing, performing ultrasonic treatment (100W, 40 KHz) for 30 min, cooling, complementing weight loss, filtering, taking the subsequent filtrate, and filtering with a 0.45 mu m microporous membrane.

The specific preparation process of the reference solution provided by the invention comprises the following steps: respectively weighing appropriate amounts of synephrine and hesperidin, precisely weighing, adding methanol to prepare control stock solutions containing 1.10 mg of synephrine and 0.335 mg of hesperidin per 1 mL respectively, precisely sucking 1 mL of the control stock solutions respectively, placing the control stock solutions in a 25mL volumetric flask, adding methanol to dilute and fix the volume, and preparing a mixed control solution containing 44.00 mu g of synephrine and 13.40 mu g of hesperidin per 1 mL.

Further, the conditions of the high performance liquid chromatography are as follows: a chromatographic column: thermo Hypersil GOLD C₁₈Chromatography column (250X 4.6 mm, 5 μm); mobile phase: acetonitrile (a) -0.1% phosphoric acid water (B); sample introduction volume: 10 mu L of the solution; flow rate: 1 mL/min; column temperature: 35 ℃; detection wavelength: 283nm, gradient elution was performed.

Further, the conditions of the gradient elution are as follows: 10-14% of A for 0-8 min; 8-17 min, 14% -16% of A; 17-38 min, 16% -18% of A; 38-50 min, 18% -30% A; 30-38% of A for 50-72 min; 72-88 min, 38% -50% A; 88-88.01 min, 50% -10% A; 88.01-98 min, 10% A.

In the quality evaluation method provided by the invention, the quality markers capable of distinguishing green tangerine orange peel from vinegar tangerine orange peel are No. 1 peak and No. 5 peak.

The invention has the beneficial effects that:

(1) the invention establishes HPLC finger prints before and after vinegar processing of green tangerine peel and carries out chemometric analysis, systematically studies the change of chemical components before and after vinegar processing of green tangerine peel, screens the quality markers of vinegar green tangerine peel, and provides scientific basis for formulating more reasonable quality standard of vinegar green tangerine peel and further studying the processing principle of vinegar processed green tangerine peel.

(2) The method for analyzing the fingerprint spectrums of the green tangerine orange peel and the vinegar green tangerine peel is stable and reliable, and can be used for researching and identifying the fingerprint spectrums of the green tangerine orange peel and the vinegar green tangerine peel. The established fingerprint spectrum analysis method can systematically reflect the difference of chemical components of crude green tangerine orange peel and vinegar green tangerine orange peel decoction pieces, and the screened new components can be used as quality markers for distinguishing the crude green tangerine orange peel and the vinegar green tangerine orange peel, so that scientific basis is provided for formulating more reasonable quality standard of the vinegar green tangerine orange peel and further researching the processing principle of the vinegar roasted green tangerine orange peel.

Drawings

FIG. 1 shows HPLC finger prints of green tangerine peel and vinegar green tangerine peel;

wherein A is green tangerine orange peel; b is vinegar green tangerine orange peel.

FIG. 2 is a HPLC chromatogram of a mixed standard.

FIG. 3 is a 3D score chart (A) and a load chart (B) of the PCA of crude pericarpium Citri Reticulatae viride and vinegar pericarpium Citri Reticulatae viride decoction pieces.

FIG. 4 is a PLS-DA model score chart (A) and a model test chart (B) of green tangerine orange peel and vinegar green tangerine orange peel.

FIG. 5 is a VIP analysis chart showing the minimum difference between crude pericarpium Citri Reticulatae viride decoction pieces and vinegar pericarpium Citri Reticulatae viride decoction pieces.

FIG. 6 is a clustering analysis dendrogram of crude pericarpium Citri Reticulatae viride decoction pieces and vinegar pericarpium Citri Reticulatae viride decoction pieces.

Detailed Description

The technical solution of the present invention is further explained and illustrated by the following specific embodiments.

Example 1

1 Instrument and reagent

1.1 instruments

Agilent 1200 HPLC (including DAD detector, Agilent USA), KQ-500DE digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.), METTLER TOLEDO one ten-thousandth electronic balance (Mettlertyol Torilat USA), and Synergy UV ultra-water purifier (Millipore USA).

1.2 reagents

Hesperidin control (batch No. 110721-; acetonitrile and methanol are in chromatographic purity; the water is self-made ultrapure water; the other reagents are analytically pure; glutinous rice aromatic vinegar (Laiyanglu vinegar food Co., Ltd., lot number: 20201222).

1.3 materials

10 batches of green tangerine peel are identified as the plant tangerine of Rutaceae by professor of Li Feng in the Chinese medicine identification and research laboratory of Shandong Chinese medicine universityCitrus reticulataProcessed fruit skin of dried young fruit or unripe fruit of Blanco and its cultivar. Sample information is shown in table 1.

Table 110 batch Green Tangerine Peel information

2.1 preparation of Citrus reticulata Blanco

Stir-frying to slight yellow according to the processing method of vinegar green tangerine peel in the first part of 2020 edition of Chinese pharmacopoeia. The numbers of the raw green tangerine orange peel decoction pieces are S1-S10, and the numbers of the vinegar green tangerine orange peel are C1-C10.

2.2 chromatographic conditions

A chromatographic column: thermo Hypersil GOLD C₁₈Chromatography column (250X 4.6 mm, 5 μm); mobile phase: acetonitrile (a) -0.1% phosphoric acid water (B); sample introduction volume: 10 mu L of the solution; flow rate: 1 mL/min; column temperature: 35 ℃; detection wavelength: 283 nm. The gradient elution conditions were: 10-14% of A for 0-8 min; 8-17 min, 14% -16% of A; 17-38 min, 16% -18% of A; 38-50 min, 18% -30% A; 30-38% of A for 50-72 min; 72-88 min, 38% -50% A; 88-88.01 min, 50% -10% A; 88.01-98 min, 10% A.

2.3 preparation of the solution

2.3.1 preparation of control solutions

Respectively weighing appropriate amount of synephrine and hesperidin, precisely weighing, and adding methanol to obtain control stock solutions containing 1.10 mg of synephrine and 0.335 mg of hesperidin per 1 mL. Precisely sucking 1 mL of the reference substance stock solution, placing the reference substance stock solution in a 25mL volumetric flask, adding methanol for dilution and fixing the volume, and preparing a mixed reference substance solution containing 44.00 microgram of synephrine and 13.40 microgram of hesperidin per 1 mL.

2.3.2 preparation of test solutions

Taking about 0.20 g of sample powder (passing through a No. 5 sieve), precisely weighing, placing in a 50 mL conical flask with a plug, precisely adding 25mL of 100% methanol, sealing, weighing, performing ultrasonic treatment (100W, 40 KHz) for 30 min, cooling, complementing weight loss, filtering, taking a subsequent filtrate, and filtering with a 0.45 mu m microporous membrane to obtain the product.

2.4 methodological considerations

2.4.1 reference Peak selection

The experiment takes hesperidin as a reference peak and is used for calculating the relative retention time and the relative peak area of a common peak of a fingerprint.

2.4.2 precision test

Taking a sample solution of raw pericarpium Citri Reticulatae viride (batch number: 2007001), continuously sampling for 6 times according to the chromatographic condition of item "2.2", recording chromatogram, introducing into the "traditional Chinese medicine chromatogram fingerprint similarity evaluation system", and recording the relative retention time of each common chromatogram peak with the retention time of the No. 3 peak (hesperidin) as the reference peak. The result shows that the relative retention time of the common peak and the RSD value range of the relative peak area are 0.04-0.97%, which indicates that the precision of the instrument meets the requirement.

2.4.3 stability test

Sampling a test solution of raw green tangerine peels (batch number: 2007001) for 0, 3, 6, 9, 12 and 24 hours respectively according to the chromatographic condition under the item of 2.2, recording a chromatogram, introducing the chromatogram into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and recording the relative retention time of each common chromatographic peak by taking the retention time of the No. 3 peak (hesperidin) as a reference. The result shows that the RSD value range of the relative retention time and the relative peak area of the common peak is 0.05-1.00%, and the test solution is stable within 24 h.

2.4.4 repeatability test

Taking raw pericarpium citri reticulatae viride (batch number: 2007001) powder, preparing a sample solution according to item '2.3.2', preparing 6 parts in parallel, injecting sample according to chromatographic condition under item '2.2', recording chromatogram, guiding the chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system, and recording relative retention time of each common chromatogram peak by taking retention time of a No. 3 peak (hesperidin) as reference. The results show that the relative retention time of the common peak and the RSD value range of the relative peak area are 0.03% -0.54%, and the method is good in repeatability.

2.5 establishment of fingerprint and analysis of similarity

2.5.1 establishment and analysis of HPLC finger prints before and after Vinegar-moxibustion of pericarpium Citri Reticulatae viride

Taking each sample, preparing a test solution according to the method under the item 2.3.2, and measuring according to the chromatographic condition under the item 2.2. Recording chromatogram, introducing software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 version A), performing Mark peak matching by using a median method and a multipoint correction method with time width of 0.10min, respectively establishing HPLC fingerprints before and after vinegar roasting of pericarpium Citri Reticulatae viride, and respectively establishing comparison maps of pericarpium Citri Reticulatae viride and vinegar pericarpium Citri Reticulatae viride as shown in figure 1. 8 common peaks are marked in the fingerprint of green tangerine orange peel, 9 common peaks are marked in the fingerprint of green vinegar orange peel, wherein the 5 th peak is a common newly increased peak in the fingerprint of green vinegar orange peel. The comparison of the control shows that the peak 1 is synephrine, the peak 3 is hesperidin and the peak 5 is an unknown component, which is shown in figure 2. The method is characterized in that a hesperidin chromatographic peak 3 with a high peak area and a good separation degree is selected as a reference peak, the relative retention time of the common peaks of all samples is basically consistent, the RSD is less than or equal to 3.21%, the difference is small, and the RSD of the relative peak area of each common peak is 25.96% -192.24%, so that the components of all batches of decoction pieces are similar, but the content of the components is different.

2.5.2 evaluation of similarity

Similarity between green tangerine orange peel and vinegar tangerine orange peel and respective reference spectra is calculated by adopting software of' A edition 2012 of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and is shown in tables 2-4. As a result, the similarity between the fingerprint of the green tangerine orange peel and the reference map is more than or equal to 0.956, the similarity between the fingerprint of the vinegar green tangerine orange peel and the reference map is more than or equal to 0.941, and the similarity between the two decoction pieces ranges from 0.937 to 0.999, so that the similarity between the samples in batches is high, the processing technology is stable, but the green tangerine orange peel and the vinegar green tangerine orange peel can not be effectively distinguished through similarity evaluation, the difference needs to be further clarified, and the two decoction pieces can be effectively distinguished.

TABLE 2 evaluation results of the similarity of fingerprints of raw pericarpium Citri Reticulatae viride decoction pieces

TABLE 3 evaluation results of similarity of fingerprint spectra of vinegar pericarpium Citri Reticulatae viride decoction pieces

TABLE 4 evaluation results of the fingerprint similarity of crude pericarpium Citri Reticulatae viride and vinegar pericarpium Citri Reticulatae viride decoction pieces

2.6 multivariate statistical analysis of fingerprint spectra of green tangerine peel and vinegar green tangerine peel

2.6.1 principal Components analysis

Introducing the area data of the common peak of the green tangerine peel and the vinegar green peel and the peak area data of No. 5 peak of the vinegar green peel into SIMCA14.1 software, establishing an unsupervised identification mode based on PCA, and observing the distribution trend of the sample, wherein the results are shown in Table 5. As can be seen from table 5, the variables can be fitted to two principal components (PC 1, PC 2), PC1 describes the sample variation of 55.8%, PC2 describes the sample variation of 27.4%, and the total predictive power is 63.1%, which is considered a better model. Green tangerine peels are gathered into one group, vinegar tangerine peels are gathered into one group, and the green tangerine peels and the vinegar tangerine peels can be effectively distinguished. The PCA three-dimensional projection graphs and the distribution graphs of 9 variables of all samples are obtained according to the principal component analysis, and the result is shown in FIG. 3. The 9 variables are distributed at different positions, reflect the proportion of each spectrum peak in the principal component analysis, and the farther the point is from the Y axis, the greater the contribution of the point to the PC1, so the contribution degree to the PC1 is from large to small: no. 8 > 9 > 6 > 2 > 3 (hesperidin) > 7 > 5 > 1 (synephrine) > 4. The farther from the X-axis the point contributes more to PC2, so the contribution to the PC2 peak is from large to small: no. 1 > No. 4 > No. 5 > No. 7 > No. 3 (hesperidin) > No. 2 > No. 9 > No. 6 > No. 8.

TABLE 5 PCA parameters

2.6.2 partial least squares discriminant analysis (PLS-DA)

PLS-DA belongs to supervised pattern recognition analysis, and in order to further screen components which have great influence on the quality of two decoction pieces, the areas of common peaks of green tangerine orange peel and vinegar green tangerine peel and No. 5 peak of vinegar green tangerine peel are taken as variables. On the basis of early-stage principal component analysis, the peak areas of two kinds of decoction pieces are respectively subjected to PLS-DA analysis by adopting SIMCA14.1 software to obtain a model score chart and a model inspection chart, and the result is shown in FIG. 4. the model score chart shows that green tangerine orange peel and vinegar tangerine orange peel can be effectively distinguished, the intersection points with the abscissa axis are all less than 0.5, the intersection points with the ordinate axis are all less than 1, and the left simulated R2 and Q2 are both less than the right side. This is done with the peak number as abscissa and the projection Value (VIP) indicating the importance of the variable as ordinate, and the result is shown in FIG. 5. The VIP value is generally considered as the main difference component of the components with the VIP value larger than 1, and the higher the VIP value is, the higher the contribution rate of distinguishing the two samples is. As can be seen from the statistical results, VIP of the peak No. 5 and the peak No. 1 is greater than 1, so that the components represented by the 2 chromatographic peaks can be considered as quality markers for distinguishing green tangerine orange peel from vinegar tangerine orange peel.

2.6.3 mean clustering analysis and hierarchical clustering analysis

The area of the peak of peak No. 5 with larger contribution value is led into SPSS Statistics 22 software for mean value K-clustering analysis, and the result is shown in Table 6. As can be seen from Table 6, the raw pericarpium Citri Reticulatae viride and vinegar pericarpium Citri Reticulatae viride can be divided into two categories, 10 batches of raw pericarpium Citri Reticulatae viride belong to group 1, most of vinegar pericarpium Citri Reticulatae viride belong to group 2, and C2, C8 and raw pericarpium Citri Reticulatae viride belong to one category. Therefore, further hierarchical clustering analysis was performed, and the results are shown in FIG. 6. As can be seen from FIG. 6, the raw green peel can be grouped together, and the vinegar green peel can be grouped together.

TABLE 6 mean K-Cluster analysis results

Through main component analysis, partial least square discriminant analysis and cluster analysis of the green tangerine orange peel and vinegar tangerine orange peel chromatograms, 2 main components exist, and the green tangerine orange peel and the vinegar tangerine orange peel can be distinguished. In order to further distinguish the difference of chemical components in the green tangerine orange peel and the vinegar green tangerine peel, a partial least square discriminant analysis model is established, the established model has better resolving capability and prediction capability, and the No. 1 peak and the No. 5 peak are determined to be the difference components of the green tangerine orange peel and the vinegar green tangerine peel. When the No. 1 peak and the No. 5 peak are jointly used or the No. 1 peak is independently used for K-clustering analysis, the distinguishing degree is not good, raw green tangerine peels and vinegar green tangerine peels cannot be distinguished, when the No. 5 peak is independently used for K-clustering analysis, the raw green tangerine peels can be gathered into one type, most of the vinegar green peels can be gathered into one type, but the mean value clustering of C2 and C8 and other vinegar green peel samples cannot be gathered into one type. The analysis reason is that the peak area of the No. 5 peak in the chromatograms of the two batches of samples is small, so that the two batches of samples cannot be effectively distinguished from the chromatogram of the raw product.

Experiments show that the peak No. 5 is a newly added chromatographic peak in all chromatograms of green tangerine orange peel of vinegar, and other newly added chromatographic peaks exist, but the newly added chromatographic peaks are different due to different samples, so the peak No. 5 is considered as a newly added component chromatographic peak after green tangerine peel is processed with vinegar.