Qualitative and quantitative analyses of chemical components of Citri Sarcodactylis Fructus from different origins based on UPLC–Q–Exactive Orbitrap–MS and GC–MS

Abstract Ultra‐high‐performance liquid chromatography–Q–Exactive Orbitrap–mass spectrometry (MS) and gas chromatography (GC)–MS were performed for the qualitative and quantitative analyses of Citri Sarcodactylis Fructus (CSF) from different origins. The contents of eight major CSF components, namely 5,7‐dimethoxycoumarin, scopoletin, hesperidin, tangeretin, nobiletin, limonin, nomilin, and stachydrine, were quantitatively analyzed. Clustering analysis and principal component analysis (PCA) were, respectively, performed to classify and compare the 10 CSF batches. One hundred and two volatile components were identified accordingly by comparing retention times, reference standards, parent peaks, fragment peaks, and findings from relevant literature. Moreover, high content of 5,7‐dimethoxycoumarin and stachydrine was detected in all the CSFs, especially in CSF‐Zhe. Therefore, the high content component coumarin “5,7‐dimethoxycoumarin” was suggested to be quality analysis component rather than hesperidin. Additionally, characteristic compounds were found to distinguish different CSFs. This work was a comprehensive study about the components of various CSF. It distinguished the basic differences in the compositions of CSF from different origins. Eventually, it provided experimental and systematic bases for the quality control analysis of CSF, which has potential application in the further research.


Based on Pharmacopoeia of the People's Republic of China (ChP),
hesperidin is the only CSF component with a content >0.030% and is therefore used in quality control. Considering that the hesperidin content of CSF is slightly low, hesperidin as a single indicator is not sufficient to distinguish CSFs from different origins. Therefore, scientific index components should be urgently explored via quantitative analysis.
Some CSF components have medicinal and commercial values (Chu et al., 2012;Yanmei et al., 2015;Zheng et al., 2021). In previous study, we have carried out a relatively comprehensive qualitative analysis of the nonvolatile components of CSF (Fu et al., 2020;Wang et al., 2021), including flavonoids, limonoids, organic acids, coumarins, and other compounds. However, the determination analysis of major pharmacological compositions and volatile oil that possesses remarkable potential value of CSFs from different regions is lacking.
Few indicators of CSF quality are confirmed. Now this study will perform further quantitative analysis and clustering analysis of the CSF major components in order to make a systematic and comprehensive study on the CSF components.
In this work, a simple, effective, and precise method was established to comprehensively determine the compositions and distinction among CSFs from different origins.

| Sample and standard preparation
For UPLC-Q-Exactive Orbitrap-MS analysis, the samples were crushed and passed through a 40-mesh sieve to obtain a small powder.

| GC-MS analysis
GC analysis was performed on a TRACE DSQ GC instrument (Thermo Finnigan, USA). TG-5SILMS GC capillary column (0.25 mm × 30 m, 0.25 μm; Thermo Fisher Scientific, USA) was applied, and the temperature program was set as follows: initially, the temperature was set at 60 ℃ for 3 min, then increased to 80 ℃ at 1 ℃·min -1 for 3 min, further increased to 250 ℃ at 10 ℃·min -1 , and finally increased to 300 ℃ at a rate of 25 ℃·min -1 for 5 min. High-purity helium, the carrier gas, was operated at a flow rate of 1.0 ml·min -1 , and 1 μl of the sample solution was injected in a splitting ratio at 50:1.

| Statistical analysis
All the data of CSF from different origins were statistically analyzed using SPSS and GraphPad Prism software. Clustering analysis and principal component analysis were applied to observe the similarities and differences of the CSFs from different origins.

| Chemical determination of CSF
Based on the previous study, the methanol extracts of 10 batches  (Table 2).

| Method validation
Good linear correlations were obtained for the eight compounds using this method with R 2 >0.995 (Table 3). The limit of detection and limit of quantification were the mass concentrations of the compound when the signal-to-noise ratios were 3 and 10, respectively.
Moreover, the relative standard deviations of the repeatability

| Concentrations of CSF components
The contents of the eight principal components in the 10 batches of CSF (Table 4)  Additionally, hesperidin concentration in CSF-Zhe was higher than those in the three other CSFs. The tangeretin contents of CSF-Zhe and CSF-Guang were relatively higher than those CSF-Chuan and CSF-Yun. However, nomilin was relatively higher in CSF-Yun than in other CSFs.
Hesperidin is a flavonoid substance that is widely found in citrus and contains substantial pharmacological activities, such as antineoplastic, anti-oxidation, and anti-inflammatory activities; its content has been detected as a biomarker for quality control in CSF as described in ChP (2020). The present study indicated that hesperidin had extraordinarily low content and low response value compared with the other main components; therefore, it is not efficient and suitable as an index for the estimation of CSF quality. In comparison, 5,7-dimethoxycoumarin has remarkable pharmacological activity (Alesiani et al., 2009;Yang & Wang, 2018) and is therefore more suitable for CSF quality control.
The contents of eight main CSF components were subjected to clustering analysis to determine the similarity among CSFs from different origins. The heat map and dendrogram of eight CSF components are shown in Figure 2. The results manifested that CSF-Zhe and CSF-Guang (S2 and S3) were classified together, and CSF-Yun and CSF-Chuan (S7) were classified together.        (Table 5). The TIC of the methanol extraction is shown in Figure 3.
To determine the difference of CSFs, principal component analysis (PCA) was performed on 35 major volatiles in the CSF samples.
Consequently, five principal components (PCs) were extracted in total, with a cumulative contribution rate of 95.48% (Table 6).   and the fourth quadrant and CSF-Guang (S2 and S3) distributed on the first quadrant. All samples of CSF-Zhe were mainly concentrated on the third quadrant and CSF-Guang (S1) distributed on the second quadrant, which indicates an obvious difference between CSF-Zhe and CSF-Guang (S1) with other origins, which is closely related to PC1. Overall, these results were in accordance with clustering analysis.

| CON CLUS IONS
In this study, a systemic qualitative and quantitative analysis of nonvolatile and volatile components was conducted. A particularly high content of stachydrine was detected in CSF, and it was the first time to performed stachydrine to quantitative analysis. A new referential index "5,7-dimethoxycoumarin" was demonstrated to assess the quality and differences among CSF samples from various origins. CSF-Zhe contained the most abundant 5,7-dimethoxycoumarin and volatile oil in the four main origins. In addition, some important alkenes in CSF-Zhe were significantly high, such as D-limonene, γterpinene, ɑ-pinene, and β-pinene, which indicated that there were certain chemical composition advantages in CSF-Zhe. From the result, tetratetracontane was found to be detected in CSF-Guang (S1), and terpinolene may be one of the characteristic compounds to distinguish CSF-Zhe from other CSFs in this study. In summary, this work provided a comprehensive analysis method in CSFs, which conveyed a better understanding and sufficient reference for their further utilization.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.