Development of the Volatile Fingerprint of Qu Aurantii Fructus by HS-GC-IMS

Volatile components are important active ingredients of Rutaceae. In this study, HS-GC-IMS (headspace-gas chromatography-ion mobility spectrometry) was used to study the volatile compounds of Qu Aurantii Fructus, and a total of 174 peaks were detected, 102 volatile organic compounds (131 peaks) were identified. To compare the volatile compounds of Qu Aurantii Fructus with its similar medical herb, Aurantii Fructus, and their common adulterants, principal component analysis (PCA) and cluster analysis (CA) were performed based on the signal intensity of all the detected peaks. The results showed that Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were clustered into one group, while their common adulterants could be well distinguished in a relatively independent space. In order to distinguish Qu Aurantii Fructus from Aurantii Fructus, the peaks other than the average intensity ±2 standard deviation (95% confidence interval) were taken as the characteristic components by using the Gallery Plot plug-in software. Additionally, the fingerprint method was established based on the characteristic compounds, which can be used to distinguish among Qu Aurantii Fructus, Aurantii Fructus and their common adulterants quickly and effectively. We found that the characteristic components with higher content of Qu Aurantii Fructus were nerol, decanal, coumarin and linalool. This study provides a novel method for rapid and effective identification of Qu Aurantii Fructus and a new dimension to recognize the relationship between Qu Aurantii Fructus and Aurantii Fructus.


Introduction
Qu Aurantii Fructus is recorded in the 2015 edition of the processing standard of traditional Chinese medicine in Zhejiang Province [1]. It is the dried, immature fruit of Citrus changshan-huyou Y.B. Chang, which is harvested in July when the fruit is still green. It has the function of regulating qi width and relieving flatulence. It is used to relieve chest and hypochondriac qi stagnation, fullness and pain, retention of food accumulation, phlegm and internal stagnation; it is often used to treat diseases such as organ ptosis. Qu Aurantii Fructus is mainly produced in Quzhou City, Zhejiang Province, which is one of the "New Zhe-ba-wei". Studies on chemical constituents show that Qu Aurantii Fructus mainly contains flavonoids [2][3][4], triterpenes [5], phenolic acids [6], steroids [6], and coumarins [2]. Modern pharmacological studies show that Qu Aurantii Fructus have pharmacological activities such as lung injury protection [7,8], liver protection [9,10], antioxidation [11], blood sugar lowering [12], anti-microbial [13], and so on.

Comparative Analysis of Unique Volatile Compounds in Different Samples
All the detected peaks of Qu Aurantii Fructus and Aurantii Fructus and their common adulterants were selected for fingerprint comparison using the Gallery Plot plug-in, as shown in Figures 3 and 4. We found that they have the same types of volatile components, but there are differences in the proportion. The unique components of different samples are shown as follows. Abbreviations: MW, Molecular weight; RI, retention index; Dt (RIP Rel.), drift time (reaction−ion−peak relative).

Comparative Analysis of Unique Volatile Compounds in Different Samples
All the detected peaks of Qu Aurantii Fructus and Aurantii Fructus and their common adulterants were selected for fingerprint comparison using the Gallery Plot plug−in, as shown in Figures 3 and 4. We found that they have the same types of volatile components, but there are differences in the proportion. The unique components of different samples are shown as follows.

Comparative Analysis of Volatile Compounds between Qu Aurantii Fructus and Aurantii Fructus
The comparison of the fingerprint profiles of Qu Aurantii Fructus and Aurantii Fructus was shown in Figure 3. It can be seen from the plot diagram that there are differences

Comparative Analysis of Unique Volatile Compounds in Different Samples
All the detected peaks of Qu Aurantii Fructus and Aurantii Fructus and their common adulterants were selected for fingerprint comparison using the Gallery Plot plug−in, as shown in Figures 3 and 4. We found that they have the same types of volatile components, but there are differences in the proportion. The unique components of different samples are shown as follows.

Comparative Analysis of Volatile Compounds between Qu Aurantii Fructus and Aurantii Fructus
The comparison of the fingerprint profiles of Qu Aurantii Fructus and Aurantii Fructus was shown in Figure 3. It can be seen from the plot diagram that there are differences

Comparative Analysis of Volatile Compounds between Qu Aurantii Fructus and Aurantii Fructus
The comparison of the fingerprint profiles of Qu Aurantii Fructus and Aurantii Fructus was shown in Figure 3. It can be seen from the plot diagram that there are differences between Qu Aurantii Fructus and some species of Aurantii Fructus. For example, the contents of citral, benzothiazole, peak 2 and 15 of Qu Aurantii Fructus are higher, the contents of hexan-2-one, pentan-1-ol and peak 9 of Citrus aurantium cv. Xiucheng are higher, the contents of hexan-2-ol, butan-2-one, linalool oxide-M and geraniol of Citrus aurantium 'Daidai' are higher, the contents of α-terpineol, vanillin, peak 13 and 25 of Citrus aurantium 'Chuluan' are higher. These differential components are the basis for the identification of Qu Aurantii Fructus and Aurantii Fructus.

Comparative Analysis of Volatile Compounds of Qu Aurantii Fructus and the Common Adulterants
Using the Gallery Plot plug-in, all the peaks of Qu Aurantii Fructus and the common adulterants were compared by fingerprint, as shown in Figure 4. The differences between the volatile compounds of Qu Aurantii Fructus and the adulterants are as follows: the relative contents of citral, benzothiazole, peak 2 and 15 are higher in Qu Aurantii Fructus; the relative contents of 3-methylbut-2-enal are higher in Citrus wilsonii Tana-ka; the relative contents of 2-oxopropyl acetate, 4-ethylphanol, (methyldisulfanyl) methane, 6-methyl-5hepten-2-one, and pentan-1-ol are higher in Citrus reticulata 'Unshiu'; the relative contents of acetophenone, 1-(furan-2-yl)ethanone, ethyl acetate and acetoin are higher in Citrus sinensis (Linn.) Osbeck. There are significant differences in volatile components between Qu Aurantii Fructus and adulterants; in particular, the three common adulterants have obvious characteristic components for identification.

Stoichiometric Analysis
To recognize the similarities and differences among Qu Aurantii Fructus, Aurantii Fructus, and their common adulterants, principal component analysis (PCA) and cluster analysis (CA) were performed based on the signal intensity of all the detected peaks; partial least square-discriminant analysis (PLS-DA) was performed to determine the contribution value of characteristic components.

Principal Component Analysis (PCA)
All the detected peaks of Qu Aurantii Fructus, Aurantii Fructus and the adulterants were imported into SIMCA-P (13.0) software for principal component analysis, as shown in Figure 5. The automatic fitting of Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were clustered into one group (I), Aurantii Fructus (Citrus aurantium 'Huangpi', Citrus aurantium cv. 'Xiucheng', Citrus aurantium 'Daidai' and Citrus aurantium 'Chuluan') were clustered into one group (II), while three common adulterants were significantly different (III). The model test showed that R 2 X was 0.953 and Q 2 was 0.820, which indicated that the model had good stability and predictability. The statistical results of PCA show that Qu Aurantii Fructus can be effectively distinguished from three kinds of adulterants, but is similar with Aurantii Fructus (Citrus aurantium L.). This is basically consistent with the same clinical efficacy of Qu Aurantii Fructus and Aurantii Fructus.

Cluster Analysis (CA) for Qu Aurantii Fructus and Aurantii Fructus
To further validate the results of PCA analysis, all the data of Qu Aurantii Fructus and Aurantii Fructus were imported into SPSS 18.0 software for cluster analysis. According to the standard, it was found that when the distance is less than 15, Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were clustered into one group, and Citrus aurantium cv. Xiucheng and Citrus aurantium 'Huangpi' were clustered into one group, as shown in Figure 6. The statistical result of CA is consistent with that of PCA.

Cluster Analysis (CA) for Qu Aurantii Fructus and Aurantii Fructus
To further validate the results of PCA analysis, all the data of Qu Aurantii Fructus and Aurantii Fructus were imported into SPSS 18.0 software for cluster analysis. According to the standard, it was found that when the distance is less than 15, Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were clustered into one group, and Citrus aurantium cv. Xiucheng and Citrus aurantium 'Huangpi' were clustered into one group, as shown in Figure 6. The statistical result of CA is consistent with that of PCA.

Cluster Analysis (CA) for Qu Aurantii Fructus and Aurantii Fructus
To further validate the results of PCA analysis, all the data of Qu Aurantii Fructus and Aurantii Fructus were imported into SPSS 18.0 software for cluster analysis. According to the standard, it was found that when the distance is less than 15, Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were clustered into one group, and Citrus aurantium cv. Xiucheng and Citrus aurantium 'Huangpi' were clustered into one group, as shown in Figure 6. The statistical result of CA is consistent with that of PCA.

Partial Least Square-Discriminant Analysis (PLS-DA)
PLS-DA was performed to determine the contribution value of characteristic components. The higher the VIP (variable importance in the projection) value of the chromatographic peak of the PLS-DA model, the greater the contribution of the chromatographic peak to the classification of the sample. The results are shown in Figure 7. Additionally, the VIP value which is greater than 1 indicates a significant effect. The results show that 23 known compounds are greater than 1.

Establishment of Characteristic Fingerprint of Qu Aurantii Fructus
The statistical results based on PCA and CA of all detected peaks showed that Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were very similar and difficult to distinguish. However, from the fingerprint profiles, there are some different components between Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.). Therefore, we tried to screen out the differential components as indicators to identify the samples.
The peaks other than the average intensity ±2 standard deviation (95% confidence interval) were taken as the characteristic components by using the Gallery Plot plug−in software, 25 characteristic compounds were screened out and fingerprints were established. As shown in Figure 8, region I is the fingerprints of different species of Aurantii Fructus, region II is the fingerprint of Qu Aurantii Fructus, and region III is the finger-

Establishment of Characteristic Fingerprint of Qu Aurantii Fructus
The statistical results based on PCA and CA of all detected peaks showed that Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were very similar and difficult to distinguish. However, from the fingerprint profiles, there are some different components between Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.). Therefore, we tried to screen out the differential components as indicators to identify the samples.
The peaks other than the average intensity ±2 standard deviation (95% confidence interval) were taken as the characteristic components by using the Gallery Plot plug-in software, 25 characteristic compounds were screened out and fingerprints were established. As shown in Figure 8, region I is the fingerprints of different species of Aurantii Fructus, region II is the fingerprint of Qu Aurantii Fructus, and region III is the fingerprints of different adulterants. It can be seen that Qu Aurantii Fructus can be distinguished among Aurantii Fructus and different adulterants, the fingerprints of Aurantii Fructus are different, and the differences are related to the varieties. Meanwhile, it can be seen from the fingerprints that the response values of nerol, decanal, coumarin and linalool are higher in Qu Aurantii Fructus, the response values of heptanal, isopentyl hexanoate, citronellol, 2-methylbutan-1-ol and coumarin are higher in Aurantii Fructus, and the response values of acetophenone, ethyl acetate, propan-1-ol, isovaleric acid and 2-methylfuran-3-thiol in adulterants are significantly higher in adulterants, which could be used as novel components to evaluate the quality of Qu Aurantii Fructus, Aurantii Fructus and the identification of the adulterants.

Discussion
In this paper, we investigated the volatile components in Qu Aurantii Fructus, Aurantii Fructus, and their common adulterants using HS−GC−IMS. Taking Aurantii Fructus as an example, by comparing the results of HS−GC−IMS with those of GC−MS analysis reported in the literatures [28,29,37], it was found that there were differences between them. The main volatile component analyzed by GC−MS was the non−characteristic component limonene, with the relative content above 50%, and the content of other volatile

Discussion
In this paper, we investigated the volatile components in Qu Aurantii Fructus, Aurantii Fructus, and their common adulterants using HS-GC-IMS. Taking Aurantii Fructus as an example, by comparing the results of HS-GC-IMS with those of GC-MS analysis reported in the literatures [28,29,37], it was found that there were differences between them. The main volatile component analyzed by GC-MS was the non-characteristic component limonene, with the relative content above 50%, and the content of other volatile components was basically below 1%. However, the volatile components analyzed by HS-GC-IMS showed that the content of limonene accounted for about 6%, linalool 7-10%, α-terpineol 4-7% and so on, the contents of more than 20 volatile components were above 1%. It is obvious that the volatile components measured by HS-GC-IMS method are more informative in terms of characteristic peaks. It is speculated that it is mainly caused by different pretreatment. When the volatile compounds are determined by GC-MS method, the sample needs steam distillation, while the sample determined by HS-GC-IMS method does not need pretreatment. The sample was grinded for direct determination, which can retain the volatile components in the sample to the maximum extent, and thus, it showed certain advantages in the identification of characteristic components.
As a similar product of Aurantii Fructus, Qu Aurantii Fructus has a very long history of use in Zhejiang Province, and its efficacy is basically the same as that of Aurantii Fructus. However, the two species currently have different legal status, as Qu Aurantii Fructus is recorded in the 2015 edition of the processing standard of traditional Chinese medicine in Zhejiang Province and can only be used in Zhejiang Province, while Aurantii Fructus is recorded in the Chinese Pharmacopoeia 2020 edition and can be used throughout China. Therefore, even if the two are similar in efficacy, they should not be mixed, and effective methods of differentiation are needed.
However, through plant taxonomic investigation, comparative study of efficacy and comparative analysis of flavonoid components [8,38,39], some scholars think that Qu Aurantii Fructus is a cultivated variety of Aurantii Fructus and can be treated without distinction.
In this paper, we compared the similarities and differences between Qu Aurantii Fructus and Aurantii Fructus in terms of volatile components, and found that they have the same types of volatile components, but there are differences in the proportion; also it was found that the volatile components in Aurantii Fructus from different sources differed significantly in the proportion. Statistical analysis (PCA and CA) was performed based on the signal intensity of all detected peaks. It was found that when the distance is less than 15, Qu Aurantii Fructus and Aurantii Fructus (Citrus aurantium L.) were clustered into one group, which showed that they have a good genetic relationship. In view of the similar clinical efficacy of Qu Aurantii Fructus and Aurantii Fructus, it is considered that a more comprehensive and in-depth study is required to examine whether Qu Aurantii Fructus can be used as a source of Aurantii Fructus.
The fingerprint was established based on the characteristic components screened by the software, which showed some specificity in the species differentiation. It can be intuitively seen from the fingerprints that the method can distinguish not only Qu Aurantii Fructus, but also different species of Aurantii Fructus, while more samples from accurate sources are needed for validation.

Materials
Eight batches of Qu Aurantii Fructus, 8 batches of Aurantii Fructus (including 2 batches of Citrus aurantium L., 1 batch of Citrus aurantium 'Huangpi', 3 batches of Citrus aurantium cv. Xiucheng, 1 batch of Citrus aurantium 'Daidai', 1 batch of Citrus aurantium 'Chuluan') and 3 batches of the common adulterants (including 1 batch of Citrus wilsonii Tana-ka, 1 batch of Citrus reticulata 'Unshiu', and 1 batch of Citrus sinensis (Linn.) Osbeck) were collected. The details of the samples are shown in Table 2. All samples were collected from their places of origin by the research group, cut in half, and dried at low temperature (40 • C).
The n-ketones C4-C9 (Sinopharm Chemical Reagent ShanghaiCo., Ltd., Shanghai, China) were used to calculate the RI of volatile compounds as external references. The drift time (RIP relative) was obtained by normalizing the drift time with the expected reaction ion peak (RIP).
Volatile compounds were identified by comparing RI and Dt (RIP Rel.) with the GC-IMS library which contains built-in NIST (National Institute of Standards and Technology, 2014) database and IMS (ion mobility spectroscopy; G.A.S; Dortmund, Germany) database. In addition, the content of each volatile compound was calculated by the normalization method based on the peak intensity.

Data Analysis
The data were acquired and analyzed using Laboratory Analytical Viewer (LAV) software and GC × IMS Library search software. LAV software includes two built-in plugins: Reporter and Gallery Plot. The Reporter plug-in was used to generate a topographic plot to visually compare the differences in 3D spectra of different samples. The Gallery Plot plug-in was used to generate fingerprint plots to visually compare the differences in peak intensities of different compounds. LAV software was used to acquire and process the IMS data and calculate the retention index (RI) of the volatile compounds using n-ketones C4-C9 as an external standard. Additionally, it was also used to filter the characteristic peaks other than the average peak intensity ±2 standard deviation (95% confidence interval) to establish the characteristic fingerprints.
Qualitative analysis was performed using GC × IMS Library search software, which contains built-in NIST (National Institute of Standards and Technology, 2014) database and IMS (ion mobility spectroscopy, G.A.S, Dortmund, Germany) database. Cluster analysis was performed by SPSS 18.0 software and principal component analysis was performed using SIMCA-P (13.0) software (MKS Data Analytics Solutions, Umea, Sweden).

Conclusions
In this study, the volatile compounds of Qu Aurantii Fructus were analyzed, and systematically compared with the components of Aurantii Fructus and their common adulterants. Based on statistical analysis, including principal component analysis (PCA) and cluster analysis (CA), the similarities and differences between Qu Aurantii Fructus and Aurantii Fructus were found. The fingerprint was established based on the characteristic components fitted by the Gallery Plot plug-in software which can be used to distinguish Qu Aurantii Fructus among Aurantii Fructus and their common adulterants effectively and quickly. The results can provide a novel reference for the quality control of Qu Aurantii Fructus and a new dimension to recognize the relationship between Qu Aurantii Fructus and Aurantii Fructus.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.