The Discrimination and Characterization of Volatile Organic Compounds in Different Areas of Zanthoxylum bungeanum Pericarps and Leaves by HS-GC-IMS and HS-SPME-GC-MS

The pericarps of Zanthoxylum bungeanum (ZBP) and leaves of Zanthoxylum bungeanum (ZBL) are popular spices in China, and they have pharmacological activities as well. In this experiment, the volatile organic compounds (VOCs) of the pericarps of Zanthoxylum bungeanum in Sichuan (SJ) and its leaves (SJY) and the pericarps of Zanthoxylum bungeanum in Shaanxi (SHJ) and its leaves (SHJY) were analyzed by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The fingerprint of HS-GC-IMS and the heat maps of HS-SPME-GC-MS were established. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed. The results showed that a total of 95 components were identified, 62 components identified by HS-SPME-GC-MS and 40 components identified by HS-GC-IMS, of which 7 were the same. The analysis found that SJ and SHJ were obviously distinguished, while SJY and SHJY were not. There were considerably fewer VOCs in the leaves than in the pericarps. In the characterization of the VOCs of ZBL and ZBP, the flavor of ZBP was more acrid and stronger, while the flavor of ZBL was lighter and slightly acrid. Thirteen and eleven differential markers were identified by HS-GC-IMS and HS-SPME-GC-MS, respectively. This is helpful in distinguishing between SHJ and SJ, which contributes to their quality evaluation.


Introduction
The pericarps of Zanthoxylum bungeanum (ZBP) are an important seasoning in China [1]. ZBP has a special flavor and is often used in Sichuan dishes. In addition to that, it is in great demand in traditional medicine [2]. Essential oils are its main active ingredients. It has anti-inflammatory [3], antiseptic [4] and other effects. Zanthoxylum bungeanum is grown in southwest China in Sichuan, Shaanxi, Yunnan and so on [5]. Its wide range of cultivation leads to different quality. It is necessary to differentiate ZBP from different regions. Aroma has an important influence on food evaluation results [6], and the volatile terpenoids are the main source of its aroma [7]. People have different opinions about ZBP from different producing areas: its color, taste, smell and even its medicinal effects. For the evaluation of the quality of pepper, experienced operators are often required. However, there is also subjectivity in judgment that affects the stability and accuracy of quality evaluation. Therefore, it is necessary to find a suitable approach to identifying it. Other than that, ZBL has special aroma. It is also used as a condiment and has some antioxidant [8] and antibacterial activity [9]. Currently, the study of volatile organic compounds (VOCs) of ZBP is abundant, but the study of VOCs of Leaves of Zanthoxylum bungeanum (ZBL) is less. It is interesting to characterize the VOCs of ZBL and investigate the similarities and differences of their composition with ZBP.
GC-MS is undoubtedly a good choice for the detection of VOCs, high sensitivity, accuracy and wide detection range, but it is a complex treatment of samples [10]. Solidphase microextraction (SPME) has the advantages of good extraction effect, selectivity, environmental friendliness and convenience [11]. The SPME combination with GC-MS can make up for some of its shortcomings, mainly the complex preprocessing steps. It was reported that SPME-GC-MS combined with chemometrics could be used to analyze the quality of strong aroma base liquor at different grades [12].
Ion mobility spectrometry (IMS), as a high-sensitivity, fast detection method, was used in the military early [13]. GC has good separation performance. By coupling GC with IMS, HS-GC-IMS ushered in a new development in food [14], cosmetics [15] and medicine [16]. In HS-GC-IMS, VOCs form into ions when they pass through the ionization source, and the ions drift in a weak electric field at atmospheric pressure; depending on the differences in structure, mass, charge and volume of each ion, the ions are detected at different times, providing information on the type and concentration of the analyte [17]. In addition, HS-GC-IMS also has the advantages of high sensitivity and environmental friendliness and no sample pretreatment [18]. Previously, it has been reported that HS-GC-IMS can identify Pericarpium Citri Reticulatae and its counterfeits [19]. GC-MS tends to be qualitative and quantitative. HS-GC-IMS tends to identify samples and can also detect small odor molecules that are not detected by GC-MS. GC-MS and HS-GC-IMS each have their own advantages. The combination of these two approaches can achieve better evaluation results [20]. It has reported that the Liuyang Douchi was determined by HS-GC-IMS and HS-SPME-GC-MS [21].
Maoxian and Hancheng are historical pepper production areas in China. Maoxian pepper has been appraised as one of China's national geographical indication products. The purpose of this study was to characterize the VOCs of ZBP of Hancheng (SHJ), ZBL of Hancheng (SHJY), ZBP of Maoxian (SJ) and ZBL of Maoxian (SJY) based on HS-GC-IMS and HS-SPME-GC-MS and investigate their differential components. Laying the foundation for the study of its active ingredients, the fingerprint of HS-GC-IMS data and the heat map of HS-SPME-GC-MS data were established. The PCA and OPLS-DA were used to distinguish the samples and find the differences in VOCs between them. Differential marker screening of ZBP and ZBL by one-way ANOVA and variable importance in projection (VIP) evaluation for the differential marker were calculated.

Materials and Chemicals
In this study, ZBP and ZBL taken from Maoxian, Sichuan, China (longitude 102.22 east latitude 31.90 north), and Hancheng, Shaanxi, China (east longitude 110.43 north latitude 35.48) ( Figure S1) were selected (for detailed information, refer to Table S1). Six batches were selected from each producing area to pick leaves and fruits in July. The N-ketone C4-C9 standard mix purchased from Sinopharm Chemical Co., Ltd. (Shanghai, China) was used to calculate retention index (RI) of HS-GC-IMS, N-alkane C8-C20 standard mix for GC-MS was purchased from Sigma-Aldrich Chemical Co., Ltd. (

HS-GC-IMS Analysis
The GC-IMS system (FlavourSpec ® , Gesellschaft für Analytische Sensorsysteme mbH, Dortmund, Germany) with an autosampler (CTC Analytics AG, Zwingen, Switzerland) was used to detect the VOCs of that ZBP and ZBL in Sichuan and Shaanxi. Weigh three samples from each batch for experiment. Weigh 0.01 g of each batch of samples and add them into a 20 mL headspace glass sampling bottle (Zhejiang HAMAG technology, Ningbo, China). Set the relevant parameters of the instrument as follows.
Conditions of HS-GC-IMS: incubation temperature was 75 • C, the incubation time was 20 min, the oscillation rate was 500 rpm, headspace injection was used, the injection temperature was 85 • C, the injection volume was 300 µL, and the carrier gas was N2 (purity ≥ 99.999%). The column was FS-SE-54-CB-1 capillary column (15 m × 0.53 mm ID, 1 µm, Beijing, China), the column temperature was 60 • C, the programmed flow was as follows: 2 mL/min initially for 2 min, increased to 10 mL/min over 10 min, increased to 150 mL/min over 20 min, and keep the flow rate of 150 mL/min over 30 min. The drift tube is 98 mm long, the temperature is 45 • C, the voltage is 500 kV, and the drift gas is nitrogen (purity ≥ 99.999%). The gas flow of drift tube was 150 mL/min.

SPME Optimization
SPME fiber (Supelco, Bellefonte, Penn.) was installed on a MultiPurpose sampler (Gerstel, GER) and combined with 7890B-7000D triple quadrupole gas chromatography mass spectrometry (Agilent Technologies, Palo Alto, CA, USA) to detect VOCs that ZBP and ZBL. Weigh three samples from each batch for experiment. weigh 0.01g for 20 mL headspace vials for each batch of samples. Incubation temperature was 75 • C, incubation time was 5 min, extraction time was 40 min, vial penetration was 21.00 mm, injection penetration was 54.00 mm desorption time was 5 min, desorption temperature was 250 • C.

Conditions of GC-MS
The chromatographic column was HP-5MS phenyl methyl siloxane (30 m × 0.25 mm × 0.25 µm, Agilent Technologies, Palo Alto, CA, USA) elastic quartz capillary column; The initial temperature was 50 • C for 2 min, then 20 • C/min to 80 • C, and 2 • C/min to 155 • C, finally 30 • C/min to 220 • C and held for 5 min; The carrier gas was Helium (purity 99.999%) with a flow rate of 1.0 mL/min; The split ratio was 1:1; The injection port temperature is 250 • C. The conditions of MS were set as follow: the ion source temperature was 230 • C, ionization energy was 70 eV with a scan range of m/z 30-650.

Statistical Analysis
Weighed an equal amount of sample as QC, each take 6 QC measured by HS-GC-IMS and HS-SPME-GC-MS, and selected 9 and 6 major components to calculate the relative standard deviation to verify the reproducibility, RSD < 10.47% and 6.67%, respectively (Table S2). Analysis of HS-GC-IMS data based on Vocal, drift time and RI were used as criteria for component identification. Analysis of GC-MS data based on Masshunter. NIST17 (match > 80, RI) as well as standard compounds were used for identification analysis. Multivariate statistical analysis by simca 14.1 (Umetrics, Malmo, Sweden). Data were normalized by peak area and normalization and scaled using pareto scaling. Plotting heat maps on MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/, accessed on 11 June 2022). Compound odor and partial activity query from ChemicalBook (www.chemicalbook.com, accessed on 15 June 2022).

HS-GC-IMS Topographic Plots in SHJY, SHJ, SJY and SJ
In HS-GC-IMS, when the concentration of components in the sample is high, the compounds will form dimers or even multimers with protons or electrons. And the presence of multimers contributes more to qualitative accuracy. The X-axis was the drift time, the Y-axis was the retention time, and the Z-axis was the ion peak intensity. The color represented the peak intensity. The peak intensity decreased from red to white and then to blue. It can be seen that most of the ion peaks appear at the retention time of 100-1600 s and the drift time of 1.0-2.5 ( Figure 1A). Identification of differences in SHJY, SHJ, SJY, SJ from three dimensions of retention time, drift time and peak intensity. The overall number of peaks of ZBP was more than that of ZBL, and the peak intensity was greater than that of ZBL. Compared with the other three samples, SHJ had two very obvious ion peaks. There was little difference in the peaks of the same part from different places, but the difference in the peak can also be seen.

HS-GC-IMS Topographic Plots in SHJY, SHJ, SJY and SJ
In HS-GC-IMS, when the concentration of components in the sample is high, the compounds will form dimers or even multimers with protons or electrons. And the presence of multimers contributes more to qualitative accuracy. The X-axis was the drift time, the Y-axis was the retention time, and the Z-axis was the ion peak intensity. The color represented the peak intensity. The peak intensity decreased from red to white and then to blue. It can be seen that most of the ion peaks appear at the retention time of 100-1600 s and the drift time of 1.0-2.5 ( Figure 1A). Identification of differences in SHJY, SHJ, SJY, SJ from three dimensions of retention time, drift time and peak intensity. The overall number of peaks of ZBP was more than that of ZBL, and the peak intensity was greater than that of ZBL. Compared with the other three samples, SHJ had two very obvious ion peaks. There was little difference in the peaks of the same part from different places, but the difference in the peak can also be seen.
A total of 62 VOCs were measured, including 1 acid, 31 alkenes, 14 alcohols, 1 phenol, 2 aldehydes, 3 ketones, 9 esters and 1 alkane (Table S3). The color indicated the logarithm of the peak area at the bottom of 10, which increased from blue to white and then to red. More VOCs of ZBP were detected by HS-SPME-GC-MS than of ZBL ( Figure 3). More (1E,4E)-germacrene B, α-bisabolol (antiinflammatory, antibacterial) were detected in SJ. In SHJ, more 4-isopropylcyclohex-2-en-1-one, cumic alcohol, cuminaldehyde (at high concentration, strong special smell of dry tea gas, which is unpleasant; sweet smell at low concentration) and so on were detected. 1,8-cineole was detected in SHJ, SJ, SJY and SHJY, but more in SHJ and SJ. More VOCs were detected in ZBP than in its leaves, such as γ-terpineol (pine-and clovelike aroma), α-terpinene, α-phellandrene (black pepper aroma and sweet lotus aroma). γ-terpineol was heavily detected in SHJ and SJ; γ-terpineol had bactericidal and anticancer potential [31]. SJY's specific VOCs were (+)-citronellal (lemon flavor, anthelmintic and antifungal). β-lonone, (-)-humulene epoxide II could be detected in SJY and SHJY. α-terpineol (clove aroma; it has strong antibacterial activity against periodontal disease and cariogenic bacteria), spathulenol, caryophyllene oxide (anti-inflammatory, anti-cancer and enhance skin permeability); all of them were detected except SJ. Terpinyl acetate (lemon and lavender fragrance), linalool (the sweet, tender and fresh flowers smell like lily of the valley), linalyl acetate (light and sweet aroma, like orange leaves, terpene-free lemon and raw pear, and like lavender flowers) were detected in all samples, and their content was higher. There was more terpinyl acetate in SHJ; more linalool and linalyl acetate in SJ, and more total ZBP than leaves.      Supplementary Table S3).

Multivariate Statistical Analysis
In statistics, too many variables can increase the complexity of the analysis. PCA is an unsupervised multivariate statistical analysis method. By comparing the principal component factors, the dimensionality of the data is reduced and regularity and difference between samples are revealed [32]. Multivariate statistics of HS-GC-SPME-MS and HS-GC-IMS were obtained for model A and model B. SHJ, SJ, SHJY and SJY were clustered in the PCA of A and B, respectively. In the PCA of model A, Q2 = 0.855, indicating that the model was good. The contribution of PC1 was 72.1%, and the contribution of PC2 was 16.5%. The positive part of PC1 could well distinguish the pericarps, and the negative part of PC1 could well distinguish the leaves. In the direction of PC2, SHJ and SJ could be distinguished ( Figure 4A). The PCA of model A was 0.924, indicating an excellent model fit. The contribution of PC1 was 62.7%; the contribution of PC2 was 34.7%; the distinction between SHJ and SJ was obvious; and the difference between SHJY and SJY was not obvious. The overall results were similar to HS-GC-IMS ( Figure 4D). Both HS-GC-IMS and HS-SPME-GC-MS can significantly distinguish between SHJ and SJ, but the distinction between ZBL of different origins is not obvious. The reason may be due to climatic and geographical factors. Maoxian is a plateau monsoon climate with sufficient sunshine, little precipitation, a dry climate and large temperature differences; Hancheng is a continental monsoon climate with mild climate, sufficient light and more rainfall. Additionally, there were relatively fewer VOCs of ZBL, which makes the difference between SHJY and SJY appear less obvious.    OPLS-DA is a supervised method for identifying differences in samples from different categories, eliminating data that are not relevant to category information [33], here, the less characteristic components of ZBL, mostly small-molecule components detected by HS-GC-IMS. To exclude the risk of overfitting, HS-GC-IMS and HS-SPME-GC-MS were cross-validated 200 times. Q2 were less than 0.05, indicated that the results are not overfitted ( Figure 4C,F). Compounds with VIP values of >1 and p < 0.05 were screened by one-way ANOVA as differential markers, and components with large errors were sieved out, the differential markers marked in red in the graph ( Figure 4G,H). The peak area is used as a reference to indicate the content, The content ranges from high to low corresponding to warm to cool tones. In HS-GC-IMS, 1,8-cineole for SHJ, neryl acetate, nerol, (E,E)-2,4-hexadienal, 4-terpineol, α-pinene, linalool oxide, acetone, β-ocimene, furaneol, αterpinene, 2-butoxyethanol for SJ, and diethyl butanedioate for SJY were differential marker ( Figure 4G). In HS-SPME-GC-MS, α-caryophyllene, linalool, linalyl acetate, DL-limonene for determining SJ, terpinyl acetate, α-thujene, piperitone, γ-terpineol, α-terpineol, sabinene for SHJ, and (+)-citronellal for CJY were differential marker ( Figure 4H).

Comprehensive Analysis
The peak areas were used as a reference for the content of the components and were analyzed for all identified components. It was discovered that the VOC content of ZBL was significantly less than that of ZBP, and the content of SHJ was slightly less than that of SJ, regardless of HS-GC-IMS or HS-SPME-GC-MS. The results of HS-SPME-GC-MS showed that SHJ has more ketones than SJ but much less esters ( Figure 5A,C). Among the VOCs detected by HS-GC-IMS, alcohols accounted for the largest proportion of both ZBL and ZBP, followed by alkenes. In addition, more abundant esters were detected in ZBP ( Figure 5B). In HS-SPME-GC-MS, more ester components were detected in all samples. In SJ, the ester component was even more than 50%. The alcohol component was less and the ester component was more than in Zanthoxylum schinifolium Sieb. et Zucc [34]. Alcohols and alkenes still account for a high proportion of all samples ( Figure 5D). Linalool, myrcene, 1,8-cineole, limonene and 3-nonanone were identified as the five predominant components [35]. A total of 95 components were identified by HS-GC-IMS and HS-SPME-GC-MS. α-Terpinene, 1.8-cineole, linalool, 4-terpineol, neryl acetate, acetic acid, α-pinene and β-pinene were the common components detected by those two approaches. HS-SPME-GC-MS detected more middle molecular alkenes, alcohols and esters, and HS-GC-IMS detected more small molecular alcohols, aldehydes and esters.

Conclusions
HS-GC-IMS and HS-SPME-GC-MS were used to characterize the VOCs of SHJY, SJY, SHJ and SJ rapidly, accurately, comprehensively and without contamination. The foundation was laid for the study of active ingredients. Moreover, the differences in VOCs were compared between SHJY, SHJ, SJY and SJ. Combined with multivariate statistics, thirteen and eleven differential markers were screened from HS-GC-IMS and HS-SPME-GC-MS, respectively, which contributed to the quality evaluation of ZBP and ZBL. Similarly, HS-GC-IMS and HS-SPME-GC-MS could also be applied to other VOCs of food, spices, traditional Chinese medicine and so on. Regrettably, the VOCs of ZBL are much less than ZBP. The flavor of ZBP is more acrid and strong, while the flavor of ZBL is lighter and slightly acrid, which is a valuable flavoring. Some active VOCs are present in ZBL and ZBP, and the results show the medical value of ZBP and ZPL. The different components of ZBP from different areas enriched the flavor of different regions but also led to its unstable efficacy. It is necessary to strictly control the origin and other factors to ensure that its components and properties are stable and controllable. In terms of quality control, HS-GC-IMS and HS-SPME-GC-MS will be good detection means.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Table S1: Detailed information of all the samples; Table S2: Reproducibility;  Table S3: HS-SPME-GC-MS integration parameters of VOCs in SHJ, SJ, SHJY, and SJY; Figure

Conclusions
HS-GC-IMS and HS-SPME-GC-MS were used to characterize the VOCs of SHJY, SJY, SHJ and SJ rapidly, accurately, comprehensively and without contamination. The foundation was laid for the study of active ingredients. Moreover, the differences in VOCs were compared between SHJY, SHJ, SJY and SJ. Combined with multivariate statistics, thirteen and eleven differential markers were screened from HS-GC-IMS and HS-SPME-GC-MS, respectively, which contributed to the quality evaluation of ZBP and ZBL. Similarly, HS-GC-IMS and HS-SPME-GC-MS could also be applied to other VOCs of food, spices, traditional Chinese medicine and so on. Regrettably, the VOCs of ZBL are much less than ZBP. The flavor of ZBP is more acrid and strong, while the flavor of ZBL is lighter and slightly acrid, which is a valuable flavoring. Some active VOCs are present in ZBL and ZBP, and the results show the medical value of ZBP and ZPL. The different components of ZBP from different areas enriched the flavor of different regions but also led to its unstable efficacy. It is necessary to strictly control the origin and other factors to ensure that its components and properties are stable and controllable. In terms of quality control, HS-GC-IMS and HS-SPME-GC-MS will be good detection means.