Rhizoma Alismatis (RA), widely known as “Ze-Xie” in China, is the tuber of
Rhizoma Alismatis (RA), widely known as “Ze-Xie” in China, is the tuber of
The main active ingredients of RA are classified into triterpenoids and sesquiterpenoids [
Many methods, such as high-performance liquid chromatography-ultraviolet detection (HPLC-UV) [
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) [
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. The 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 [
Thus, both sesquiterpenoids and triterpenoids are considered necessary for the quantitative analysis in RA. This 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.
Reference standards of RA alismoxide, alisol C, alisol C 23-acetate, alisol A, alisol A 24-acetate, alisol B, 11-deoxyalisol B, and 11-deoxyalisol B 23-acetate were purchased from Chengdu Mansite Biotechnology Co., Ltd. (Chengdu, China). Alisol B 23-acetate was purchased from National Institutes for Food and Drug Control (Beijing, China). Alismol was purchased from Yunnan Xi Li Biotechnology Co., Ltd. (Yunnan, China). The 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. The 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
Chemical structures of the 10 investigated compounds and 1 internal standard.
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). The sampling method was based on different wilt states. The collection dates and codes were according to GAP conducted by Nanping Institute of Agricultural Sciences of Fujian Province, as follows (Figure
Different wilt stages of RA. (a) Stage I. (b) Stage II. (c) Stage III. (d) Stage IV. (e) Stage V. (f) Stage VI.
On the other hand, a total of 42 batches of baked samples (processing temperature research) were collected on February 23, 2017. Each of the seven processing groups (freeze dryer, 60°C, 70°C, 80°C, 100°C, 120°C, and 150°C) weighed 30.0 kg (fresh RA) and was randomly divided into six parallels. Then, the fresh RA was placed in a freeze dryer, and others were dried in a heat pump oven at 60°C (60°C–1, 60°C–2, 60°C–3, 60°C–4, 60°C–5, 60°C–6), 70°C (70°C–1, 70°C–2, 70°C–3, 70°C–4, 70°C–5, 70°C–6), 80°C (80°C–1, 80°C–2, 80°C–3, 80°C–4, 80°C–5, 80°C–6), 100°C (100°C–1, 100°C–2, 100°C–3, 100°C–4, 100°C–5, 100°C–6), 120°C (120°C–1, 120°C–2, 120°C–3, 120°C–4, 120°C–5, 120°C–6), and 150°C (150°C–1, 150°C–2, 150°C–3, 150°C–4, 150°C–5, 150°C–6).
All the RA materials were authenticated as tuber of
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.
The 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. The extracted solution was centrifuged at 12 000 rpm for 10 min. The supernatant was obtained as a sample solution. A total of 500
The UPLC-MS/MS analysis was performed with an ACQUITY UHPLC I-Class system (Waters, Milford, MA, USA) coupled with Xevo TQ–S tandem quadrupole mass spectrometer (Waters, Milford, MA, USA). Data acquisition and quantification were conducted with MassLynx version 4.1 data software (Waters, MA, USA). Chromatographic separation was carried out at 45°C on Waters CORTECS C18 column (2.1 mm × 100 mm, 1.6
For the calibration curves, at least ten concentrations of calibration standard solution were made and analysed in triplicate. Then, the calibration curve of each analyte was constructed from the peak area ratios of each standard to IS against the concentration of each analyte. The 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.
The 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. The 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. The percentage recoveries were calculated according to the following equation: (detected amount − original amount) × 100%/spiked amount. The RSD was used to evaluate the results.
Different methods were compared to achieve extraction efficiency. The following methods were tested: extraction methods (e.g., ultrasound, reflux, soxhlet, and warm immersion), extraction solvents (e.g., 40%, 60%, 80%, and 100% acetonitrile), extraction time (e.g., 15, 30, 45, and 60 min), and sample-to-solvent ratio (e.g., 1 : 50, 1 : 100, 1 : 125, and 1 : 150). The optimal sample preparation was the extraction of 0.2 g sample with 25 mL of 100% acetonitrile in an ultrasonic water bath for 30 min (Supplementary Materials, Figure
Factors (column, mobile phase, and column temperature) that affected the separation of multicomponent sample were optimised to achieve the simultaneous separation of triterpenoids and sesquiterpenoids. After the comparison of ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7
For triterpenoids, the ESI-MS spectra were acquired in the multiple reaction monitoring (MRM) mode with a positive electrospray ion source (ESI+). The MRM product ion, collision energy, cone voltage, ion pairs, and the details of the proposed fragmentation pathway of each compound were systematically optimised (Supplementary Materials, Figures
The APCI-MS (Figure
Optimisation of multiple reaction monitoring product ions, APCI-MS/MS spectra and the proposed fragmentation pathway of alismoxide (a) and alismol (b).
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
Retention time, related MS data of the 10 investigated compounds, and internal standards in the UPLC-APCI/ESI-MS/MS analysis.
Compounds |
|
Fragment ions ( |
Cone voltage (V) | Collision energy (eV) | Ionisation modes |
---|---|---|---|---|---|
Alismoxide | 1.48 | 203.1 ⟶ 161.0; |
15 | 15 | APCI+ |
Alisol C | 2.05 |
|
35 | 15 | ESI+ |
Alisol C 23-acetate | 2.57 | 529.3 ⟶ 511.3; 529.3 ⟶ 469.3; |
30 | 20 | ESI+ |
Alisol A | 3.03 | 473.3 ⟶ 337.0; 473.3 ⟶ 365.3; |
40 | 11 | ESI+ |
Alisol A 24-acetate | 3.5 |
|
20 | 10 | ESI+ |
Alismol | 3.64 | 203.1 ⟶ 161.0; |
10 | 12 | APCI+ |
Alisol B | 4.23 | 455.1 ⟶ 437.0; 455.1 ⟶ 383.1; 455.1 ⟶ 379.1; |
40 | 27 | ESI+ |
Alisol B 23-acetate | 4.99 | 497.1 ⟶ 479.1; |
40 | 8 | ESI+ |
11-Deoxyalisol B | 5.81 |
|
40 | 10 | ESI+ |
11-Deoxyalisol B 23-acetate | 6.68 |
|
35 | 12 | ESI+ |
Glycyrrhetnic acid (IS) | 3.76 | 417.3 ⟶ 317.2 | 35 | 12 | ESI+ |
∗Quantitative ion pair.
UPLC-ESI/APCI-MS/MS chromatogram of 10 target standards and 1 internal standard of standard solution (a) and RA sample (b). AX: alismoxide; C: alisol C; 23C: alisol C 23-acetate; A: alisol A; 24A: alisol A 24-acetate; AL: alismol; B: alisol B; 23B: alisol B 23-acetate; 11-B: 11-deoxyalisol B; 11-23B: 11-deoxyalisol B 23-acetate.
The UPLC-MS/MS method was validated with precision, linearity, lower limit of quantification (LOQs), lower limit of detection (LODs), repeatability, stability, and recovery.
The 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. The 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
Regression equation, LODs, and LOQs of the 10 investigated compounds.
Compound | Regression equations | Linear range (ng·mL−1) |
|
LODs (ng·ml−1) | LOQs (ng·ml−1) |
---|---|---|---|---|---|
Alismoxide |
|
1.021–1021 | 0.9999 | 0.16 | 0.51 |
Alisol C |
|
4.928–492.8 | 0.9998 | 0.82 | 1.64 |
Alisol C 23-acetate |
|
5.105–5105 | 0.9997 | 0.73 | 2.55 |
Alisol A |
|
0.872–872.0 | 0.9999 | 0.14 | 0.44 |
Alisol A 24-acetate |
|
3.12–3120 | 0.9990 | 0.51 | 1.25 |
Alismol |
|
3.00–3000 | 0.9998 | 0.48 | 1.51 |
Alisol B |
|
7.728–7728 | 0.9985 | 0.62 | 3.86 |
Alisol B 23-acetate |
|
11.3–11300 | 0.9971 | 1.67 | 5.65 |
11-Deoxyalisol B |
|
3.432–3432 | 0.9985 | 0.46 | 1.72 |
11-Deoxyalisol B 23-acetate |
|
5.348–5348 | 0.9997 | 0.93 | 1.18 |
The 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. The solutions for the interday precision test were examined, for 3 days. The 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
Six RA samples (Stage IV–6) were extracted and analysed to confirm their repeatability. The RSD values of 10 analytes were within the range of 0.60%–2.02%. Stability sample solution was analysed within 24 h (0, 2, 4, 8, 12, and 24 h) at room temperature to investigate their stability. Repeatability and stability for real samples were less than 2.78% and 3.19% within 24 h, respectively (Supplementary Materials, Table
Recovery was used to evaluate the accuracy of the method and determine by adding standard solutions with three different concentration levels (low, medium, and high) to the known amounts of RA sample (
Table
Table
Contents of the 10 investigated compounds in 36 batches of RA harvesting samples. AX: alismoxide, C alisol C 23C: alisol C 23-acetate, A alisol A 24A: alisol A 24-acetate, AL: alismol, B alisol B 23B: alisol B 23-acetate, 11-B: 11-deoxyalisol B 11-23B: 11-deoxyalisol B 23-acetate.
Sample code | AX | C | 23C | A | 24A | AL | B | 23B | 11-B | 11-23B |
---|---|---|---|---|---|---|---|---|---|---|
Stage I–1 | 0.022 | 0.153 | 0.288 | 0.022 | 0.013 | 0.172 | 0.310 | 0.538 | 0.094 | 0.037 |
Stage I–2 | 0.017 | 0.164 | 0.345 | 0.025 | 0.017 | 0.158 | 0.290 | 0.698 | 0.083 | 0.070 |
Stage I–3 | 0.017 | 0.195 | 0.329 | 0.018 | 0.012 | 0.129 | 0.257 | 0.845 | 0.093 | 0.056 |
Stage I–4 | 0.025 | 0.174 | 0.351 | 0.028 | 0.010 | 0.212 | 0.293 | 0.749 | 0.110 | 0.083 |
Stage I–5 | 0.015 | 0.152 | 0.318 | 0.021 | 0.015 | 0.284 | 0.266 | 0.865 | 0.097 | 0.090 |
Stage I–6 | 0.021 | 0.173 | 0.323 | 0.017 | 0.015 | 0.200 | 0.284 | 0.752 | 0.126 | 0.046 |
|
||||||||||
Stage II–1 | 0.025 | 0.135 | 0.246 | 0.020 | 0.021 | 0.216 | 0.285 | 0.966 | 0.116 | 0.043 |
Stage II–2 | 0.018 | 0.122 | 0.335 | 0.020 | 0.020 | 0.218 | 0.246 | 0.831 | 0.100 | 0.057 |
Stage II–3 | 0.022 | 0.119 | 0.314 | 0.027 | 0.020 | 0.236 | 0.307 | 0.829 | 0.105 | 0.046 |
Stage II–4 | 0.023 | 0.120 | 0.276 | 0.028 | 0.027 | 0.239 | 0.330 | 0.883 | 0.116 | 0.084 |
Stage II–5 | 0.028 | 0.143 | 0.303 | 0.022 | 0.024 | 0.205 | 0.291 | 0.762 | 0.123 | 0.115 |
Stage II–6 | 0.022 | 0.147 | 0.288 | 0.022 | 0.024 | 0.217 | 0.352 | 0.767 | 0.132 | 0.063 |
|
||||||||||
Stage III–1 | 0.030 | 0.139 | 0.238 | 0.029 | 0.026 | 0.226 | 0.326 | 0.747 | 0.104 | 0.066 |
Stage III–2 | 0.032 | 0.128 | 0.285 | 0.035 | 0.039 | 0.222 | 0.303 | 0.718 | 0.099 | 0.059 |
Stage III–3 | 0.034 | 0.111 | 0.238 | 0.028 | 0.031 | 0.210 | 0.267 | 0.802 | 0.088 | 0.064 |
Stage III–4 | 0.033 | 0.123 | 0.216 | 0.030 | 0.030 | 0.219 | 0.301 | 1.049 | 0.104 | 0.077 |
Stage III–5 | 0.034 | 0.094 | 0.291 | 0.029 | 0.034 | 0.231 | 0.326 | 0.924 | 0.090 | 0.108 |
Stage III–6 | 0.026 | 0.123 | 0.225 | 0.027 | 0.034 | 0.197 | 0.360 | 0.998 | 0.104 | 0.063 |
|
||||||||||
Stage IV–1 | 0.029 | 0.090 | 0.181 | 0.053 | 0.077 | 0.252 | 0.319 | 1.034 | 0.136 | 0.041 |
Stage IV–2 | 0.037 | 0.087 | 0.199 | 0.064 | 0.081 | 0.287 | 0.319 | 0.999 | 0.106 | 0.059 |
Stage IV–3 | 0.031 | 0.088 | 0.160 | 0.055 | 0.070 | 0.271 | 0.363 | 0.993 | 0.137 | 0.094 |
Stage IV–4 | 0.033 | 0.084 | 0.182 | 0.058 | 0.075 | 0.266 | 0.494 | 1.253 | 0.175 | 0.091 |
Stage IV–5 | 0.038 | 0.097 | 0.157 | 0.054 | 0.062 | 0.313 | 0.344 | 1.201 | 0.154 | 0.113 |
Stage IV–6 | 0.036 | 0.092 | 0.170 | 0.062 | 0.068 | 0.260 | 0.368 | 1.078 | 0.134 | 0.069 |
|
||||||||||
Stage V–1 | 0.029 | 0.071 | 0.150 | 0.033 | 0.034 | 0.376 | 0.634 | 1.488 | 0.216 | 0.057 |
Stage V–2 | 0.030 | 0.081 | 0.173 | 0.031 | 0.038 | 0.412 | 0.610 | 1.477 | 0.195 | 0.062 |
Stage V–3 | 0.028 | 0.073 | 0.159 | 0.039 | 0.037 | 0.423 | 0.674 | 1.399 | 0.230 | 0.066 |
Stage V–4 | 0.032 | 0.084 | 0.156 | 0.028 | 0.041 | 0.379 | 0.639 | 1.460 | 0.252 | 0.108 |
Stage V–5 | 0.028 | 0.083 | 0.146 | 0.032 | 0.031 | 0.402 | 0.565 | 1.544 | 0.234 | 0.121 |
Stage V–6 | 0.032 | 0.080 | 0.159 | 0.036 | 0.033 | 0.433 | 0.607 | 1.538 | 0.211 | 0.073 |
|
||||||||||
Stage VI–1 | 0.029 | 0.131 | 0.204 | 0.036 | 0.028 | 0.315 | 0.439 | 0.742 | 0.165 | 0.047 |
Stage VI–2 | 0.036 | 0.124 | 0.222 | 0.043 | 0.032 | 0.434 | 0.363 | 0.819 | 0.193 | 0.067 |
Stage VI–3 | 0.035 | 0.102 | 0.235 | 0.031 | 0.024 | 0.457 | 0.343 | 0.828 | 0.186 | 0.081 |
Stage VI–4 | 0.027 | 0.134 | 0.193 | 0.035 | 0.034 | 0.428 | 0.357 | 0.701 | 0.169 | 0.098 |
Stage VI–5 | 0.032 | 0.105 | 0.177 | 0.031 | 0.031 | 0.517 | 0.362 | 0.882 | 0.196 | 0.103 |
Stage VI–6 | 0.030 | 0.100 | 0.204 | 0.035 | 0.030 | 0.445 | 0.436 | 0.949 | 0.208 | 0.066 |
Broken line diagram of the contents of 10 compounds in RA harvesting samples (a). Variation histogram of the total compounds in RA harvesting samples in 6 stages (b). Variation histogram of total triterpenoids and two sesquiterpenoids in the harvesting samples in 6 stages (c). AX: alismoxide, C: alisol C 23C: alisol C 23-acetate, A alisol A 24A: alisol A 24-acetate, AL: alismol, B alisol B 23B: alisol B 23-acetate, 11-B: 11-deoxyalisol B 11-23B: 11-deoxyalisol B 23-acetate.
Table
Contents of the 10 investigated compounds in 42 batches of different RA dry temperature samples. AX: alismoxide, C alisol C 23C: alisol C 23-acetate, A alisol A 24A: alisol A 24-acetate, AL: alismol, B alisol B 23B: alisol B 23-acetate, 11-B: 11-deoxyalisol B 11-23B: 11-deoxyalisol B 23-acetate.
Sample no. | AX | C | 23C | A | 24A | AL | B | 23B | 11-B | 11-23B |
---|---|---|---|---|---|---|---|---|---|---|
Freeze-dried–1 | 0.030 | 0.074 | 0.146 | 0.033 | 0.034 | 0.393 | 0.641 | 1.489 | 0.230 | 0.097 |
Freeze-dried–2 | 0.030 | 0.078 | 0.179 | 0.031 | 0.039 | 0.440 | 0.626 | 1.473 | 0.201 | 0.120 |
Freeze-dried–3 | 0.029 | 0.085 | 0.166 | 0.039 | 0.040 | 0.391 | 0.673 | 1.420 | 0.232 | 0.114 |
Freeze-dried–4 | 0.032 | 0.072 | 0.163 | 0.032 | 0.044 | 0.382 | 0.632 | 1.472 | 0.242 | 0.115 |
Freeze-dried–5 | 0.028 | 0.084 | 0.150 | 0.033 | 0.035 | 0.430 | 0.567 | 1.580 | 0.242 | 0.125 |
Freeze-dried–6 | 0.032 | 0.080 | 0.161 | 0.037 | 0.034 | 0.439 | 0.619 | 1.549 | 0.220 | 0.109 |
|
||||||||||
60°C–1 | 0.026 | 0.069 | 0.163 | 0.042 | 0.045 | 0.362 | 0.588 | 1.413 | 0.211 | 0.093 |
60°C–2 | 0.028 | 0.071 | 0.169 | 0.039 | 0.046 | 0.417 | 0.596 | 1.323 | 0.200 | 0.103 |
60°C–3 | 0.025 | 0.059 | 0.163 | 0.037 | 0.046 | 0.403 | 0.620 | 1.477 | 0.205 | 0.096 |
60°C–4 | 0.026 | 0.057 | 0.150 | 0.031 | 0.044 | 0.356 | 0.577 | 1.339 | 0.216 | 0.101 |
60°C–5 | 0.028 | 0.063 | 0.156 | 0.047 | 0.050 | 0.396 | 0.548 | 1.412 | 0.195 | 0.110 |
60°C–6 | 0.024 | 0.060 | 0.163 | 0.049 | 0.041 | 0.393 | 0.571 | 1.381 | 0.177 | 0.099 |
|
||||||||||
70°C–1 | 0.025 | 0.055 | 0.143 | 0.045 | 0.055 | 0.341 | 0.516 | 1.179 | 0.198 | 0.089 |
70°C–2 | 0.027 | 0.050 | 0.154 | 0.046 | 0.050 | 0.367 | 0.564 | 1.374 | 0.206 | 0.086 |
70°C–3 | 0.024 | 0.055 | 0.156 | 0.046 | 0.042 | 0.377 | 0.544 | 1.248 | 0.186 | 0.081 |
70°C–4 | 0.025 | 0.055 | 0.148 | 0.044 | 0.049 | 0.347 | 0.570 | 1.160 | 0.197 | 0.089 |
70°C–5 | 0.024 | 0.049 | 0.143 | 0.050 | 0.055 | 0.372 | 0.548 | 1.265 | 0.174 | 0.094 |
70°C–6 | 0.024 | 0.050 | 0.154 | 0.051 | 0.044 | 0.361 | 0.528 | 1.184 | 0.190 | 0.079 |
|
||||||||||
80°C–1 | 0.020 | 0.037 | 0.113 | 0.085 | 0.113 | 0.303 | 0.411 | 0.881 | 0.150 | 0.064 |
80°C–2 | 0.019 | 0.046 | 0.128 | 0.080 | 0.143 | 0.268 | 0.456 | 0.933 | 0.139 | 0.068 |
80°C–3 | 0.018 | 0.044 | 0.117 | 0.082 | 0.140 | 0.304 | 0.430 | 1.017 | 0.127 | 0.073 |
80°C–4 | 0.016 | 0.046 | 0.120 | 0.099 | 0.116 | 0.309 | 0.491 | 0.856 | 0.131 | 0.062 |
80°C–5 | 0.019 | 0.039 | 0.104 | 0.075 | 0.129 | 0.296 | 0.435 | 0.823 | 0.141 | 0.060 |
80°C–6 | 0.021 | 0.043 | 0.121 | 0.074 | 0.120 | 0.272 | 0.474 | 0.806 | 0.140 | 0.057 |
|
||||||||||
100°C–1 | 0.019 | 0.039 | 0.088 | 0.131 | 0.165 | 0.245 | 0.376 | 0.706 | 0.119 | 0.063 |
100°C–2 | 0.018 | 0.035 | 0.083 | 0.119 | 0.185 | 0.272 | 0.305 | 0.782 | 0.107 | 0.071 |
100°C–3 | 0.017 | 0.038 | 0.092 | 0.121 | 0.190 | 0.241 | 0.377 | 0.830 | 0.103 | 0.067 |
100°C–4 | 0.015 | 0.040 | 0.085 | 0.138 | 0.166 | 0.251 | 0.362 | 0.720 | 0.115 | 0.059 |
100°C–5 | 0.018 | 0.035 | 0.069 | 0.101 | 0.179 | 0.233 | 0.402 | 0.763 | 0.092 | 0.052 |
100°C–6 | 0.020 | 0.039 | 0.092 | 0.121 | 0.157 | 0.221 | 0.349 | 0.836 | 0.101 | 0.055 |
|
||||||||||
120°C–1 | 0.019 | 0.032 | 0.062 | 0.116 | 0.317 | 0.193 | 0.286 | 0.566 | 0.092 | 0.060 |
120°C–2 | 0.018 | 0.027 | 0.078 | 0.159 | 0.296 | 0.234 | 0.314 | 0.616 | 0.088 | 0.056 |
120°C–3 | 0.020 | 0.035 | 0.071 | 0.151 | 0.302 | 0.196 | 0.275 | 0.570 | 0.079 | 0.047 |
120°C–4 | 0.021 | 0.025 | 0.083 | 0.139 | 0.360 | 0.213 | 0.292 | 0.648 | 0.095 | 0.063 |
120°C–5 | 0.014 | 0.029 | 0.088 | 0.128 | 0.292 | 0.189 | 0.339 | 0.695 | 0.081 | 0.049 |
120°C–6 | 0.019 | 0.037 | 0.062 | 0.136 | 0.353 | 0.199 | 0.282 | 0.622 | 0.082 | 0.050 |
|
||||||||||
150°C–1 | 0.012 | 0.028 | 0.051 | 0.169 | 0.471 | 0.137 | 0.227 | 0.522 | 0.054 | 0.048 |
150°C–2 | 0.010 | 0.017 | 0.070 | 0.190 | 0.423 | 0.172 | 0.241 | 0.477 | 0.067 | 0.054 |
150°C–3 | 0.013 | 0.012 | 0.053 | 0.153 | 0.473 | 0.165 | 0.171 | 0.505 | 0.058 | 0.045 |
150°C–4 | 0.010 | 0.028 | 0.050 | 0.161 | 0.521 | 0.148 | 0.225 | 0.427 | 0.051 | 0.048 |
150°C–5 | 0.008 | 0.022 | 0.064 | 0.149 | 0.401 | 0.168 | 0.206 | 0.375 | 0.065 | 0.052 |
150°C–6 | 0.011 | 0.029 | 0.060 | 0.141 | 0.460 | 0.157 | 0.221 | 0.523 | 0.049 | 0.048 |
Different processing temperature of RA.
Broken line diagram of the contents of 10 compounds in different RA dry temperature samples (a). Variation histogram of the total compounds in different RA dry temperature samples (b). AX: alismoxide, C: alisol C 23C: alisol C 23-acetate, A alisol A 24A: alisol A 24-acetate, AL: alismol, B alisol B 23B: alisol B 23-acetate, 11-B: 11-deoxyalisol B 11-23B: 11-deoxyalisol B 23-acetate.
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. Then, 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.
The data used to support the findings of this study are included within the article and supplementary information file.
The authors declare that they have no conflicts of interest.
This work was supported by the National Natural Science Foundation of China (81872990, 81703692, and 81673561), National Chinese Herbal Medicine Standardisation Project (ZYBZH-Y-FJ-09), Youth Backbone Talent Project of Fujian Provincial Health Commision (2018-ZQN-65), Young Researcher Project of Fujian Provincial Health Commision (2016-2-29), Subject of Fujian Province Science and Technology Hall of China (2018J01870), Key Platform Project of Fujian University of Traditional Chinese Medicine (X2017009), and the Fujian Province College Students’ Innovative Entrepreneurial Training Plan Project (201610393030). The authors thank Fujian Chengtian Pharmaceutical Co., Ltd for providing RA samples.
Figures S1–S11 and Table S1–S4 show the comprehensive analysis.