Quantitative Determination of p-Cymene, Thymol, Neryl Acetate, and β-Caryophyllene in Different Growth Periods and Parts of Eupatorium fortunei Turcz. by GC-MS/MS

Eupatorium fortunei Turcz. is a widely used Chinese herbal medicine in China. In this study, a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) method was developed and validated to simultaneously determine the contents of p-cymene, thymol, neryl acetate, and β-caryophyllene in roots, stems, and leaves of Eupatorium fortunei Turcz. harvested at different growth periods. All four constituents could be detected in leaves, three could be detected in stems except β-caryophyllene, and only thymol could be detected in roots. The order of the total contents of four constituents in different parts was leaves > stems > roots. It indicated that the leaves could be the proper medicinal parts of Eupatorium fortunei Turcz. The content of four constituents in leaves varied a lot among different growth periods and showed an M-shaped change trend with the growth of Eupatorium fortunei Turcz. The four constituents accumulated to the highest values in early July followed by mid-September. Accordingly, the best harvest time of Eupatorium fortunei Turcz. is early July and mid-September.


Introduction
Eupatorium fortunei Turcz., one species of the genus Eupatorium (Compositae), is a commonly used aromatic traditional herbal medicine in China. It has been applied for relieving nausea, vomiting, and poor appetite due to dampness obstruction since thousands of years ago [1]. Previous studies showed that the plant contains a number of bioactive natural products [2,3]. e content determination of total volatile oil in Eupatorium fortunei Turcz. is stipulated in Chinese Pharmacopeia, and the total volatile oil is consequently used as an important index to evaluate its quality [4], while various studies showed that constituents in the volatile oil of Eupatorium fortunei Turcz. from different regions and harvest time varied a lot [5][6][7]. Accordingly, it is of great significance to assay representative constituents in the volatile oil of Eupatorium fortunei Turcz.
Based on the related literature, p-cymene and neryl acetate were the earliest separated and identified compounds from the volatile oil of Eupatorium fortunei Turcz. [8]. It has been reported that p-cymene possesses an effect on dispelling phlegm [9]. Together with neryl acetate, it was also claimed to be the main active ingredient of the volatile oil for the treatment of influenza [10]. β-Caryophyllene was reported to be the constituent with the highest content in the volatile oil and has been studied as an indicator constituent to establish the content determination method of Eupatorium fortunei Turcz. [11,12]. ymol and its derivatives are abundant in the plant [2,[13][14][15]. It could be concluded that the contents of p-cymene, thymol, neryl acetate, and β-caryophyllene (structured as shown in Figure 1) have a significant effect on the quality of Eupatorium fortunei Turcz.
It is well known that traditional Chinese medicines have the characteristic of complex composition. e composition and content of compounds are closely related to the quality of traditional Chinese medicine. e growth period is always an important factor affecting the composition and content of constituents in the medicinal herbs. us, the appropriate harvesting time for traditional Chinese medicine is crucial to the formation of high-quality traditional Chinese medicine [16]. A lot of works have been performed on the effect of the developmental stage on the content of biologically active compounds in plants [17][18][19]. Accordingly, the quantities of the volatile oil in Eupatorium fortunei Turcz. may change during the growth stages. It is essential to study the content change of the volatile oil with growth for ensuring the quality of Eupatorium fortunei Turcz. to prevent and treat disease. erefore, in this work, Eupatorium fortunei Turcz. was harvested at different growth periods, and the contents of pcymene, thymol, neryl acetate, and β-caryophyllene in different parts were determined by using GC-MS/MS. e dynamic characteristic and distribution of four constituents were analyzed.

Plant Materials. Eupatorium fortunei
Turcz. was collected every fifteen days from April 15 to November 1, 2019, at the medicinal botanical garden of Xi'an (34°25' N, 108°98' E) and identified by Prof. Xiaofeng Niu. Ten plants were harvested at each stage. Each plant was separated into leaves, stems, and roots. en, the leaves, stems, and roots were separately collected together and dried.

GC-MS/MS Analysis.
A Shimadzu GC-MS 8040 equipped with SH-Rxi-5Sil MS capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness) and triple quadrupole mass spectrometer was used (Japan). e helium of high purity (99.9999%) was the carrier gas at a flow rate of 1 mL·min −1 . Samples were injected in a split mode with a ratio of 10 : 1. e temperature procedure of the column was set at 70°C for 3 min, increased to 80°C at a rate of 10°C·min −1 , to 100°C at a rate of 20°C·min −1 , to 130°C at a rate of 2°C·min −1 , and to 280°C at a rate of 20°C·min −1 , and maintained for 2 min. An electron ionization system was used with ionization energy of 70 eV. Injection, interface, and ionization temperature were set at 250°C, 250°C, and 230°C, respectively. We used standard compounds of each volatile under the MRM mode to assay the contents of four target components [20].

Preparation of Standard Solution and Samples.
Each standard stock solution was prepared separately by dissolving accurate amount of compound in hexane. A series of working solutions of these 4 compounds were freshly prepared by diluting the mixed standard solution with hexane.
Dried leaves, stems, and roots of Eupatorium fortunei Turcz. were milled to powder by using a high-speed disintegrator, and 0.2 g ground powder was extracted with 20 mL hexane in an ultrasonic waterbath for 60 min. Additional hexane was added to make up the lost weight [16].
en, the solution was filtered through a 0.22 μm filter before GC-MS/MS analysis.

Statistical Analysis.
All measurements were performed using triplicate samples. Data were expressed as mean ± SD. All data were statistically processed using GraphPad Prism 7 (GraphPad Software, CA, USA) and analyzed by Tukey's multiple comparisons test to calculate the difference significance at a 5% level (p < 0.05).

Optimization of GC-MS Analysis Conditions and Method
Validation. Temperature procedure, parameters of ion pairs, and collision voltage are the key parameters for the GC-MS/MS method. ese parameters were optimized in the present study. MRM was used for quantitative purposes with one target ion pair and two reference ion pairs. e corresponding collision voltage was optimized and is listed in Table 1. e MRM chromatogram is shown in Figure 2.

Optimization of Sample Process Conditions.
e influence factors, including extraction solvent, method, and duration were investigated through single-factor experiments. Extraction solvents including ethanol, ethyl acetate, petroleum ether (60°C∼90°C), and hexane were compared, and hexane resulted in a preferable peak and baseline. Two classical extraction methods, ultrasonic extraction, and reflux, were investigated. e ultrasonic extraction showed better extraction efficiency. en, we set the extraction duration at 15 min, 30 min, 45 min, and 60 min. e contents of compounds were increased with the prolongation of extraction time (Table 3). erefore, the extraction was conducted by using hexane in an ultrasonic waterbath for 60 min.
All the constituents could be detected in leaves, and only thymol exists in roots. e order of the contents of four constituents in different parts was leaves > stems > roots. Volatile oil exists in glandular hairs, oil rooms, oil tubes, secretory cells, or resin ducts. Volatile oil exists in different parts of different plants. Some of them are found in the whole plant, while others are found in some organs of flowers, fruits, leaves, roots, or rhizomes. e location of volatile oil varies with plant species. Hence, one can see that the studied four compounds mainly existed in leaves of Eupatorium fortunei Turcz. Higher content of essential oil in leaf than other organs of a plant has been previously reported for other species [19,23]. Based on that, it could be concluded that the leaves should be the best medicinal part of Eupatorium fortunei Turcz.

Dynamic Characteristics of Four Constituents in Different Growth Periods of Eupatorium fortunei Turcz.
e concentrations change of p-cymene, thymol, neryl acetate, and β-caryophyllene in different parts of Eupatorium fortunei Turcz. with growth time were also analyzed. e contents of all constituents in leaf showed a trend of increase-decrease-increase-decrease with the plant growing.
e content of p-cymene accumulated to its highest value on July 15. After an 80% reduction, the content rose again to 86% of the highest value approximately on September 15.
ymol reached its first peak value on August 15 and then reached the highest level on September 15. e highest value of thymol was about 6.5 times and 3.3 times higher than that on September 1 and October 1. e content of neryl acetate was the highest from July 1 to July 15, and it was also high on September 15, which is about 74% of the highest value. e yield of β-caryophyllene was the highest on July 1 and then decreased sharply. e content of β-caryophyllene increased again from 17.86 μg·g −1 on September 1 to 44.38 μg·g −1 on September 15. e contents of four constituents in the stem at each harvest stage showed different patterns. e yield of p-cymene was the highest on August 15. After a 63% reduction, p-cymene content rose again to a higher value on October 15. While the content of thymol was highest on April 15, it then decreased with the plant growth. Similarly, the content of neryl acetate reached the highest value on April 15 and had the second highest content on July 15, which was about 90% of the highest value. Only thymol could be detected in roots from June 1 to August 15, and the content was the highest on June 1.
In summary, the contents of four constituents in different parts of Eupatorium fortunei Turcz. reached the highest value in early July, followed by mid-September. ese two time points could be considered as the best time to harvest the plant.
To the best of our knowledge, there are no studies regarding the relationship between the growth stages of Eupatorium fortunei Turcz. and the content of essential oil extracted from it. In other studies, it has been found that the contents of many biological active compounds in plants depend on growth time; the yields of compounds usually reach the highest at summer or flowering stages (July or August) [18,19].  Journal of Analytical Methods in Chemistry

Conclusions
A quantitative determination method was developed and validated to assay the contents of p-cymene, thymol, neryl acetate, and β-caryophyllene in Eupatorium fortunei Turcz. by GC-MS/MS. e contents of the four constituents in samples from different parts were assayed and distribution was analyzed. All constituents could be detected in leaves. e contents order of constituents in different parts was leaves > stems > roots. From that, we could deduce that leaves should be the best medicinal part of Eupatorium fortunei Turcz. e growth stages of the plant strongly influence the content of four analytes in Eupatorium fortunei Turcz.
e four constituents accumulated to the highest value in early July followed by mid-September. Accordingly, the best harvest time of Eupatorium fortunei Turcz. is early July and mid-September.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Guanjun Nan and Lina Zhang contributed equally to this work.