Qualitative and Quantitative Analysis of Major Triterpenoids in Alismatis Rhizoma by High Performance Liquid Chromatography/Diode-Array Detector/Quadrupole-Time-of-Flight Mass Spectrometry and Ultra-Performance Liquid Chromatography/Triple Quadrupole Mass Spectrometry

Alismatis Rhizoma (AMR) is a well-known natural medicine with a long history in Chinese medicine and has been commonly used for treating a wide range of ailments related to dysuria, edema, nephropathy, hyperlipidaemia, diabetes, inflammation as well as tumors in clinical applications. Most beneficial effects of AMR are attributed to the presence of protostane terpenoids, the major active ingredients of Alismatis Rhizoma (AMR). In this study, a systematic high performance liquid chromatography/diode-array detector/quadrupole-time-of-flight mass spectrometry (HPLC-DAD-Q-TOF MS) and ultra-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC-QqQ MS) method was developed for qualitative and quantitative analyses of the major AMR triterpenoids. First, a total of 25 triterpenoid components, including 24 known compounds and one new compound were identified by comparison with UV spectra, molecular ions and fragmentation behaviors of reference standards or the literature. Second, an efficient method was established for the rapid simultaneous determination of 14 representative triterpenoids by UPLC-QqQ MS. Forty-three batches of AMR were analyzed with linearity (r, 0.9980–0.9999), intra-day precision (RSD, 1.18%–3.79%), inter-day precision (RSD, 1.53%–3.96%), stability (RSD, 1.32%–3.97%), repeatability (RSD, 2.21%–4.25%), and recovery (98.11%–103.8%). These results indicated that new approaches combining HPLC-DAD-Q-TOF MS and UPLC-QqQ MS are applicable in the qualitative and quantitative analysis of AMR.


Introduction
Alismatis Rhizoma (AMR) are dried rhizomes of Alisma orientale (Sam.) Juzep., it is a well-known natural medicine with long history in Chinese medicine. As a traditional medicine in China, AMR is an important part of many prescriptions and has been commonly used for treating a wide range of ailments related to dysuria, edema, nephropathy, hyperlipidaemia, diabetes, inflammation as well as tumors in clinical applications [1][2][3][4][5]. Most beneficial effects of AMR are attributed to the presence of protostane terpenoids, which are relatively abundant in this preparation. Previous studies have indicated that triterpenoids have diverse biological activities such as hypolipidemic [1], anti-proliferative activities [6], antibacterial [7], antiplasmodial [8], antioxidative [9] and anti-inflammatory bioactivities [10].
Previously, multiple studies have reported the analysis of triterpenes in AMR by LC-UV, LC-ELSD and LC-MS [11][12][13]. However, they focused on characterizing the limited compounds, with shortcomings such as long analysis time and poor LOQs [11,12], sometimes providing only qualitative data [13]. To the best of our knowledge, few studies assessing the systematic chemical profile and quantification of AMR were reported. Therefore, it is of great significance to develop a method for qualitative and quantitative analyses of AMR chemical constituents, which would be beneficial to studies evaluating AMR efficacy and quality.
Recently, liquid chromatography-mass spectrometry (LC-MS) offers the possibility to obtain a more comprehensive chemical profile and quantitative by utilizing multiple ionization techniques and/or different ion modes [14][15][16]. LC coupled with the time-of-flight (TOF) mass spectrometry, it has the capability and advantage to produce exact mass measurements, which provides the elemental compositions of unknown peaks with high accuracy. It also provides data on accurate precursor and/or product ions with high accuracy (routinely below 5 ppm), which substantially enhances the metabolite characterization reliability [17,18]. Beside, simultaneous quantification of multi-components has been widely performed in the analysis of traditional Chinese medicine (TCM) [19]. The ultra-performance liquid chromatography (UPLC) method has become one of the most frequently applied approaches in the area of fast chromatographic separations. Moreover, triple quadrupole mass spectrometry (QqQ MS) has higher sensitivity than ELSD and UV detections, especially for the non-UV-absorbing such as triterpenoids present in AMR. UPLC coupled with triple quadrupole mass spectrometry (UPLC QqQ MS) with high sensitivity and effectiveness provides a reliable quantification of multi-components in TCM [20,21].

Optimization of HPLC-DAD-Q-TOF MS Conditions
For qualitative analysis, in order to improve HPLC resolution and sensitivity, and shorten the analytical time, variables such as column type, column temperature, mobile phase, and flow rate can be optimized. We found that use of Ultimate XB ODS-C18 column (4.6 × 150 mm, 5 μm, Welch, Concord, MA, USA) results in improved peak capacity, stronger retention ability, and better resolution compared with other columns; therefore, it was selected for the study. Different mobile phases, including methanol-water, methanol-water (containing 0.1% formic acid), acetonitrile-water and acetonitrile-water (containing 0.1% formic acid) were examined. Interestingly, sharp peaks were achieved with acetonitrile-water (containing 0.1% formic acid) which proved to be the suitable mobile phase due to the good resolution and mass spectrum response for the most of the analytes. Meanwhile, the effects of column temperature (25,30,35, and 40 °C) and flow rate (0.6, 0.8, 1.0 mL/min) were also studied. Finally, column temperature of 30 °C and flow rate of 0.8 mL/min were optimal for qualitative analysis. Likewise, TOF MS parameters, including ion modes, capillary voltage, ion modes and collision energy (CE) were also optimized.

Optimization of UPLC-QqQ MS Conditions
For quantitative, UPLC mobile phases, including water-methanol, water-acetonitrile, methanol-water (containing 0.1% formic acid), and acetonitrile-water (containing 0.1% formic acid) were examined to obtain optimal chromatograms. As a result, good analyte separation was achieved with acetonitrile-water (containing 0.1% formic acid). In addition, the most appropriate precursor ion, daughter ion, cone voltage, collision energy (CE) were adjusted according to each analyte. Finally, the most sensitive transitions in MRM were selected. Glycyrrhetinic acid was chosen as internal standard due to the similar structure, retention time, and ionization response in ESI-MS. The MS data of fourteen related analytes are shown in Table 1.
Type VI: Type VI compounds showed a unique 13,17-oxide ring and produced a typical ion at m/z 399.2896. Their maximum UV absorption was below 200 nm due to the absence of conjugated bonds. For example ( Figure 4F Compounds 8, 12, and 19 which showed similar fragmentation behaviors to 13,17-epoxy-alisol A, were identified as 13,17-epoxy-alisol A 24-acetate, 13,17-epoxy-alisol B and 13,17-epoxy-alisol B 23-acetate, respectively. All were further confirmed by comparison with their reference standards.
Type VII: Compounds 7 and 13 produced a typical ion at m/z 399.2896, just like type VI triterpenoids. Compared to type VI compounds, they showed an unsaturated bond at C-11-C-13, which resulted in maximum UV absorption at 245 nm (λmax = 245 nm). Compound 7 (an example, Figure 4G

Method Validation
The linear calibration curves were produced by plotting the ratios of the peak areas of each standard to IS against the concentration of each analyte. Acceptable linear correlation in these conditions was confirmed by correlation coefficients (r, 0.998 0-0.999 9). The LODs (S/N = 3) and LOQs (S/N = 10) for the 14 standard analytes were 1.01-9.23 and 3.91-27.4 ng/mL, respectively, indicating that this method is sensitive for the quantitative determination of major components in AMR samples (Table 3). The RSD values of intra-day and inter-day variations, repeatability and stability of the target components were 1.18%-3.79%, 1.53%-3.96%, 2.21%-4.25%, and 1.32%-3.97% respectively. The recovery rate of the fourteen standards varied from 98.11% to 103.8% (RSD ≤ 4.06%). These results are summarized in Table 4. In conclusion, the developed method had good linearity, precision, repeatability, stability, and accuracy. Table 4. Precision, repeatability, stability, and recovery of fourteen analytes (n = 6).

Analytes
Precision for Standard Solution (n = 6) Repeatability from Real Samples Stability (%) Accuracy

Sample Analysis
This developed analytical method was successfully applied to simultaneously determine the fourteen major components in forty-three AMR batches obtained from two major GAP bases. The UPLC-QqQ MS MRM chromatograms in the positive ion mode of the 14 components are shown in Figure 5. Their contents are summarized in Table 5. The results indicated different contents for these triterpenes in crude drugs from different origins. Among these compounds, alisol B and alisol B 23-acetate were dominant compounds in all samples, at amounts of 0.104-1.232 mg/g and 1.131-2.032 mg/g, respectively. Comparing the crude drugs, obvious differences could be observed between Fujian and Sichuan samples: 16-oxo-alisol A 23-acetate, 16-oxo-alisol A 24-acetate, alisol C, alisol F, alisol A, alisol A 23-acetate, alisol A 24-acetate, alisol G and alisol B were found at higher amounts in Sichuan samples but 11-doxy-alisol B was lower compared with Fujian samples. And the other compounds, including alisol C 23-acetate, alisol L, alisol F 24-acetate and alisol B 23-acetate showed no significant difference between the two sources ( Figure 5).

Materials and Standards, Reagents
Forty-three batches of AMR samples were collected from two major good agricultural practice (GAP) bases approved by State Food and Drug Administration of China (SFDA). These crude drugs were identified by Prof. Shui-Sheng Wu. Voucher specimens were deposited in the College of Pharmacy, Fujian University of Traditional Chinese Medicine.

Preparation of Standard Solution and Samples
All standards were individually dissolved in acetonitrile to approx. 1 mg/mL. The stock solution for each quantitative analyte was further diluted with acetonitrile to achieve a series of working solutions used to establish the calibration curves. All solutions were stored at 4 °C, and filtered through a 0.22 μm membrane before use.
Forty-three batches of AMR samples were ground to fine powder and well mixed. Exactly 0.20 g powder was weighted and ultrasonicated for 30 min in 25 mL acetonitrile. The extraction solution was centrifuged at 16,000× g for 10 min, and the supernatant filtered through a 0.22 μm membrane for analysis.
The internal standard glycyrrhetinic acid was prepared in acetonitrile to a final concentration of about 0.5 μg/mL. 500 μL of this working solution were added to 500 μL of each sample solution or mixed standard solution, vortexed and filtered through a 0.22 μm membrane before analysis.
Mass spectra were acquired in the positive ion mode with electrospray ionization (ESI) source. The ESI-MS condition was optimized: capillary voltage, 3.5 kV; nebulizer pressure, 2.0 bar; dry gas (N2) flow rate, 4 L/min; dry gas temperature, 180 °C; spectrum rate, 1.7 Hz; scan range, m/z 50-1000; funnel 1 and 2, 200.0 Vpp; hexapole Rf, 120.0 Vpp; quadrupole ion energy, 3.0 eV; collision Rf, 350.0 Vpp. Argon was used as the collision gas, with collision energy set at 10-50 eV to obtain ion fragment data. External instrument calibration was applied daily before sample analysis in order to achieve an acceptable accuracy threshold at 5 ppm. Accurate mass data of the molecular ions were processed by the Data Analysis software (Bruker Daltonics, Bremen, Germany).The preparative high-performance liquid chromatography coupled with a mass spectrometer detector auto-purification system (Waters, Manchester, UK), including a Waters 2545 apparatus equipped with a 2767 fraction collector, a Waters SQD2 quadrupole mass spectrometer, and a Waters Xbridge C18 (19 mm × 150 mm, 5 μm, Waters) column, was used for preparation.
Mass spectrometry was performed on tandem mass spectrometer with an electrospray ionization (ESI) source. Nitrogen was used as nebulizer, curtain and heater gas; Argon was used as collision gas. The nebulizergas was set at 500 L/h at 200 °C in the positive ion MRM mode. The cone gas was used at a flow rate of 50 L/h, with the source temperature set at 150 °C. The capillary voltage was 3.0 kV. Most proper cone voltageand collision energy (CE) were selected according to each analyte. UPLC-QqQ MS MRM chromatogram in positive ion mode of (a) fourteen target standards and (b) sample of AMR in Figure 6.