Quantitative Analysis of Eight Triterpenoids and Two Sesquiterpenoids in Rhizoma Alismatis by Using UPLC-ESI/APCI-MS/MS and Its Application to Optimisation of Best Harvest Time and Crude Processing Temperature

Rhizoma Alismatis (RA), widely known as “Ze-Xie” in China, is the tuber of Alisma orientale (Sam.) Juzep (Alismaceae), a Chinese herbal medicine that has been used to treat hyperlipidemia, diabetes, hypertension, dysuria, and inflammation. In this study, a sensitive and reliable method based on an ultra-performance liquid chromatography (UPLC) couple with two ionisation modes, including electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry (MS), namely, UPLC-ESI/APCI-MS/MS was developed and validated to simultaneously determine 8 triterpenoids (ESI mode) and 2 sesquiterpenoids (APCI mode) in RA. Ten marker compounds were analysed with a Waters' CORTECS UPLC C18 column (200 mm × 2.1 m, 1.6 μm) and gradient elution with water (contained 0.1% formic) and acetonitrile within 7 min. The established method was validated for linearity, intra- and interday precisions, accuracy, recovery, and stability. The calibration curve for 10 marker compounds showed good linear regression (r > 0.9971). The limits of detection and quantification for analytes were 0.14–1.67 ng/mL and 0.44–5.65 ng/mL, respectively. The relative standard deviations (RSD, %) and accuracy (RE, %) of intra- and interday precisions were less than 3.83% and 1.21% and 3.22% and 1.46%, repeatability and stability for real samples were less than 2.78% and 3.19%, respectively. All recoveries of the 10 marker compounds ranged from 97.24% to 102.49% with RSDs less than 4.05%. The developed method efficiently determined the 10 marker compounds in RA and was subsequently applied to optimise harvest time and crude processing temperature. The result indicated the 90% wilted phase and 70°C (or lower) may be the best harvest time and the processing temperature of RA.

Many methods, such as high-performance liquid chromatography-ultraviolet detection (HPLC-UV) [19], high-performance liquid chromatography with an evaporative light scattering detector (HPLC-ELSD) [20], ultraperformance liquid chromatography (UPLC) [21], highperformance liquid chromatography/diode-array detector/quadrupole-time-of-flight mass spectrometry (HPLC-DAD-Q-TOF MS) [22], ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF MS) [23], and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) [24], have been established for the qualitative and quantitative analyses of RA for triterpenoid. Gas chromatography-mass spectrometry (GC-MS) was used in the qualitative analysis of RA for the volatile oil, containing sesquiterpenoids [25]. Nevertheless, these methods are only used for the qualitative or quantitative analysis of one type terpenoid (triterpenoid or sesquiterpenoid). To the best of our knowledge, simultaneous detection of triterpenoids and sesquiterpenoids in RA by UPLC-MS/MS have not been previously reported, which are limited to understand the distribution of two kinds of active terpenoid, especially in different harvest times or the processing temperature of RA samples.
APCI and ESI are two ionisation methods in the MS source. APCI is used to analyse small molecule compounds with medium polarity and some volatile compounds. ESI is used to analyse polar compounds and biomacromolecules (nonvolatile) [17]. So, liquid chromatography coupled with two ionisation modes, electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry, namely, UPLC-ESI/APCI-MS, is suitable for the analysis of both triterpenoids and sesquiterpenoids in RA.
On the other hand, in contrast to western medicine, traditional Chinese medicine (TCM) has the characteristics of complex composition (multiple types of chemical components) and low poisonousness. e appropriate harvesting and processing technologies for TCM are crucial to the formation of high-quality TCM. Processing method is the key to the retention of chemical components [26][27][28]. A folk saying states, "in the season is medicine, after the season is grass" and "march harvesting is Artemisia capillaris unb., April harvesting is Artemisia annua L., and harvesting in May and June is firewood," such as Apocynum venetum L. leaves [29] and Artemisia annua L. [30]. erefore, determining the appropriate time for drug collection is the key to ensuring the quality of medicinal materials. is step directly affects the efficiency of medicinal materials for disease prevention and treatment. However, although some articles investigated the harvesting or processing of RA [31][32][33][34][35], they focused only on triterpenoids such as alisol B 23-acetate or/and alisol A 24-acetate (few involved sesquiterpenoids).
us, the content accumulation process of the two categories (sesquiterpenoids and triterpenoids) in the growth process and the processing of RA was not fully characterised.
us, both sesquiterpenoids and triterpenoids are considered necessary for the quantitative analysis in RA. is work aims to develop an UPLC-ESI/APCI-MS/MS for simultaneous determination triterpenoids and sesquiterpenoids in RA and subsequently apply it to optimise the harvest time and crude processing temperature of RA. . e purity of each standard was higher than 98% by using HPLC-UV and their structures were confirmed by NMR. LC-MS grade acetonitrile (Merck (Darmstadt, Germany)) and formic acid (Sigma-Aldrich, St Louis, MO, USA) were used for chromatographic optimisation. e ultrapure water (18 MΩ/cm) was obtained from Millipore Milli-Q water purification system (Millipore, Bedford, USA). All other reagents were at least of analytical purity and commercially available. Figure 1 shows the chemical structures of these compounds.

Materials and Methods
A total of 36 batches of harvest season-fresh RA samples were collected at different time points in Nanping, a Good Agricultural Practices (GAP) planting base of RA in Fujian Province by SFDA, China (established in 2001). e sampling method was based on different wilt states. e collection dates and codes were according to GAP conducted by Nanping Institute of Agricultural Sciences of Fujian Province, as follows ( Figure 2): Stage I: no wilted, Stage II: 10% wilted, Stage III: 30% wilted, Stage IV: 50% wilted, Stage V: 90% wilted, Stage VI: wilted, sprout regeneration again; Six positions were randomly selected on the same sampling field. At least 0.5 kg of fresh RA was collected per location and wilt states. e husk and fibrous roots of fresh RA were removed and dried under 45°C.
All the RA materials were authenticated as tuber of Alisma orientale (Sam.) Juzep by medicinal botanist Fan Shi-ming (School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122), and the voucher specimens were deposited in the School of Pharmacy, Fujian University of Traditional Chinese Medicine. All samples were powdered to a homogeneous size (80 mesh) prior their use.

Preparation of Standard Solution and Samples.
Each standard stock solution was prepared separately by dissolving accurate amount of compound in acetonitrile. A series of working solutions of these 10 analytes were freshly prepared by diluting the mixed standard solution with acetonitrile at the ratios of 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/mL. An internal standard stock solution was also prepared in a concentration of 400 ng/mL for glycyrrhetinic acid. All solutions were stored at 4°C before analysis.
e RA samples had a total of 78 batches (including 36 batches of harvest season samples and 42 batches of baked samples). 0.20 g powder was accurately weighted and extracted with 25 mL acetonitrile in an ultrasonic bath (50 kHz, 300 W) for 30 min. Additional acetonitrile was added to make up the lost weight. e extracted solution was centrifuged at 12 000 rpm for 10 min. e supernatant was obtained as a sample solution. A total of 500 μL of the internal standard working solution was added to 500 μL of the mixed standard or sample solution; then, the vortex was blended for 1 min and filtered through a 0.22 μm micropore membrane prior to injection. All the samples were stored at 4°C before analysis. Journal of Analytical Methods in Chemistry water was set as the mobile phase A and acetonitrile was set as the mobile phase B. A gradient elution was used as follows: 46%-46% B at 0-0.5 min, 46%-65% B at 0.5-1 min, 65%-90% B at 1-5 min, 90%-100% B at 5-6 min, 100%-46% B at 6-6.1 min, and 46%-46% B at 6.1-7.0 min. e flow rate was 0.25 mL/min, and the sample volume injected was 2 μL. Mass spectrometer conditions were optimised as follows: desolvent gas temperature, 180°C; capillary voltage, 3.5 kV; source temperature, 150°C; desolvent gas flow, 800 L/h; and cone gas flow, 150 L/h. Dwell time was set at 20 ms.

Validation of Quantitative Method
2.4.1. Linearity, LOQs, and LODs. For the calibration curves, at least ten concentrations of calibration standard solution were made and analysed in triplicate. en, the calibration curve of each analyte was constructed from the peak area ratios of each standard to IS against the concentration of each analyte. e standard solution with the lowest concentration was further diluted to a certain concentration to evaluate the LODs (S/N ratio of 3) and LOQs (S/N ratio of 10), respectively.

Precision, Repeatability, Stability, and Accuracy.
e analysis of intra-and interday precisions was carried out by six repetitive injections of a mixed standard solution in the same day and three consecutive days, respectively. Both assays were determined by performing three different concentration levels and LOQs of the standards.
Six RA samples (Stage IV-6) were prepared independently to check the repeatability. To investigate the stability, Stage IV-6 sample solution was analysed within 24 h (0, 2, 4, 8, 12, and 24 h) at room temperature. e recovery was used to evaluate the accuracy of the method and determine by adding the standard solutions with three different concentration levels (low, medium, and high) to the known amounts of RA sample. e percentage recoveries were calculated according to the following equation: (detected amount − original amount) × 100%/spiked amount.

Optimisation of UPLC
Retention time, related MS data of the 10 investigated compounds, and internal standards in the UPLC-ESI/APCI-MS/MS analysis were summarized and are shown in Table 1. Figure 4 shows the optimised MRM chromatogram of the 10 markers.

Linearity, LOQs, and LODs.
e calibration curves, which were plotted with at least ten concentrations of standard solutions, were constructed from the peak area ratios of each standard to IS against the concentration of each analyte. e LODs (S/N � 3) and LOQs (S/N � 10) for the 10 standard analytes were in the range of 0.14-1.67 ng/mL and 0.44-5.65 ng/ mL, indicating that this method is sensitive for the quantitative analysis in this study (Table 2).  Journal of Analytical Methods in Chemistry

Precision.
e precision of the developed method was determined on the basis of intra-and interday variations. For the intraday precision test, the standard solutions were analysed six times, and three different concentrations and LOQs were used in a single day. e solutions for the interday precision test were examined, for 3 days. e relative standard deviations (RSD%) and accuracy (RE%) of intra-and interday precisions were less than 3.83%, 1.21%, and 3.22%, 1.46%, respectively (Supplementary Materials, Table S2).  recoveries � (detected amount − original amount) × 100% spiked amount .

Repeatability and
(1) (RSDs ≤ 4.05%), thereby presenting the acceptable recovery and accuracy of this method. Table 3 shows the quantification results of these compounds in the 36 batches of RA from different harvest times (different wilt    I II III IV V VI  I II III IV V VI   I II III IV V VI  I II III Figure 2). Accumulation of 10 analysis compounds is shown in Figure 5. At the beginning of the growth, the contents, such as alisol B, alisol B 23-acetate, 11-deoxyalisol B, and 11-deoxyalisol B 23-acetate metabolites, were low in no wilted state at the initiation stage. Subsequently, the contents rapidly increased to the highest level in the 90% wilted state and then decreased at the stage of wilted, sprout regeneration. For alisol A and alisol A 24-acetate, the content of 50% wilted stage reached the highest level and then decreased. For alisol C and alisol C 23-acetate, the changing trend was almost opposite to alisol Bs, that is, the content of 90% wilted state reached the lowest level. In general, the content of alismoxide and alismol is constantly accumulating during the growth of RA. Combining triterpenoids with sesquiterpenoids, the results showed that sample time of the 90% wilted stage had the highest amount of total compounds (3.180 mg/g). According to the traditional harvesting time of RA [1], it usually occurred in wilted RA, and the result indicated the 90% wilted stage may be the best.

Optimisation of the Best Harvest Time.
3.6. Optimise the Best Processing Temperature. Table 4 shows the quantification results of the 42 batch samples ( Figure 6) of RA from different dry temperatures. e results (Figure 7) showed that the contents of alismoxide, alisol C, alisol C 23-acetate, alismol, alisol B, alisol B 23-acetate, 11deoxyalisol B, and 11-deoxyalisol B 23-acetate decreased with the baking temperature. Total contents were decreased with the baking temperature increased, especially when the temperature is above 80°C. Meanwhile, it is obvious that the contents alisol A and alisol A 24-acetate began to generate, and its content is increased with the temperature rise. Analysis of total contents suggested that retention ratio of the total contents is rapidly reduced from 93.4% to 68.8% (lower than 80%) when dried under 80°C, the chemical composition is obviously destroyed by high temperature. When the temperature reaches 150°C, the content of the index component alisol B 23-acetate drops below 0.5 mg/g, the appearance and content of the index component do not meet the requirements of the China Pharmacopoeia. So, we suggested the processing temperature setted at 70°C or lower.

Conclusions
In summary, a UPLC-ESI/APCI-MS/MS method for simultaneous determination of eight triterpenoids and two sesquiterpenoids in RA has been developed and validated for the first time. MS spectra were acquired in the MRM mode with APCI, and ESI was specifically used for the determination of sesquiterpenoids and triterpenoids, respectively. en, it is successfully applied to the optimal best harvest time and crude processing temperature to provide basis for the production and processing of RA, the result indicated the 90% wilted phase may be the best harvest time and the processing temperature suggested at 70°C or lower.

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

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