Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis

G Model

ARTICLE IN PRESS

MASPEC-15745; No. of Pages 10

International Journal of Mass Spectrometry xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms

Young Scientist Feature

Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis Xinglong Chen a,b , Changan Geng a,c , Xiuqiong Zhang a,b , Tianze Li a,c , Jijun Chen a,c,∗ a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China c Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming 650201, PR China

a r t i c l e

i n f o

Article history: Received 12 October 2016 Received in revised form 12 December 2016 Accepted 10 January 2017 Available online xxx Keywords: LCMS-IT-TOF Global identification strategy Diagnostic ions Protostane triterpenoids Alisma orientalis

a b s t r a c t The liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) combined with a global identification strategy was applied to studying the protostane triterpenoids (PTs) in the rhizomes of Alisma orientalis in this paper. Initially, the MSn analyses of four reference compounds were performed to summarize the fragmentation rules of PTs. Subsequently, a series of ions of PTs were presented by LC–MSn analyses of the crude extract of alismatis rhizome, from which six ions at m/z 339, 353, 365, 381, 383 and 397 with high frequency of occurrence were selected as diagnostic ions to classify the peaks. Twenty peaks were classified into six families based on common ions. A network was established for the six families via diagnostic ions. 20 PTs including 1 new compound and 19 known ones were characterized through such strategy, fragmentation rules and references. Ultimately, the detected new compound and two known compounds were isolated from the total extract of A. orientalis under the guide of above method, of which the new compound was identified as 11-deoxylalisol F by MS and NMR spectrum. This systematical investigation on the PTs demonstrated that such a strategy could be widely used in the analysis and separation of natural products. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Alismatis rhizoma, the rhizome of A. orientalis, is a famous Chinese traditional herb distributed in the south of China which is used as diuretic, hypolipidemic and hypoglycemic agent as well as anti-cancer drug in clinical applications [1]. The main chemical components of alismatis rhizoma were elucidated as protostane triterpenoids and sequiterpenes in previous reports [2]. PTs were considered as the most significant constituents of alismatis rhizoma with wide bioactivities such as diuretic effect [3], antihypertension [4], antihyperglycemic activity [5], antitumor [6] and regulation of 5-HT3A receptors [7]. In addition, we found that PTs from alismatis rhizoma presented good antihepatitis B virus activity and synthesized a series of PTs derivatives to reveal their structure-activity relationships [2,8–10]. So far, a number of analytical methods were developed for detecting and identifying PTs including high

∗ Correspondence to: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, No. 132 Lanhei Road, Kunming 650201, PR China. E-mail address: [email protected] (J. Chen).

performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) [11], ion trap mass spectrometry (HPLC/IT-MS) [12] and quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF-MS) [13]. However, the reported means could not establish a systematic method for analyzing PTs in alismatis rhizoma due to the limitation of selectivity or sensitivity. Therefore, it is necessary to develop and build a global and systematic method to profile the PTs in alismatis rhizoma. Compared with traditional analytical tools, liquid chromatography with ion trap time-of-flight tandem mass spectrometry (LCMS-IT-TOF) presented the chromatographic and mass spectral information at the same time with high resolution and sensitivity which is regarded as a powerful means in natural products analyses. Our previous investigation on the chemical compounds of S. guangxiense by LCMS-IT-TOF led to the detection and characterization of 50 compounds [14]. We also studied the fragmentation pathways of four C19 diterpenoid alkaloids and six C21 steroidal aglycones by LCMS-IT-TOF which provided useful information to understand their fragmentation behaviors [15,16]. Avula et al. used LCMS-IT-TOF to identify and characterize the C21 steroidal glycosides in Hoodia gordonii and proposed a generalized fragmentation pathway of C21 steroidal glycosides [17]. Nevertheless, the reported

http://dx.doi.org/10.1016/j.ijms.2017.01.004 1387-3806/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model MASPEC-15745; No. of Pages 10

ARTICLE IN PRESS X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

2

endeavors involving in the use of LCMS-IT-TOF in natural products analyses were largely dependent on reference compounds or the comparisons. The detection and identification of the complex constituents in natural materials was dissatisfactory in view of the difficulties in obtaining reference compounds or the comparisons. Hao et al. came up with a generally applicable strategy to achieve the goal of global detection and identification of components from herbal preparation. The developed method included three steps: the determination of diagnostic ions, the establishment of a network based on family classification and database querying. This strategy came from the idea that the natural products could usually be classified into different families, and a certain family of the components shared the common ions in mass spectrometry. Once, these common ions of the components in a certain family were confirmed. Then, the components in the same family were structurally characterized, the chemical structures in other families could also be profiled further corresponding to the established network based on these common ions. Hao et al. used this novel approach to research the components in Mai-Luo-Ning injection and identified 87 compounds [18]. Thus LCMS-IT-TOF combined with the global identification strategy is practical to establish a rapid analytical method to recognize constituents in natural materials. In this paper, LCMS-IT-TOF combined with the global identification strategy was used to study the PTs in alismatis rhizoma. 20 PTs including 1 new compound and 19 known ones were characterized through such strategy. The new compound and two known compounds were isolated from the total extract of alismatis rhizoma under the guide of above method. The structures were identified by MS and NMR spectrum. This is the first time to utilize LCMSIT-TOF and the global identification strategy to study the PTs in alismatis rhizoma. 2. Experimental section 2.1. LCMS-IT-TOF and the global identification strategy Firstly, four reference compounds were analyzed by MSn to summarize the fragmentation rules of PTs. Then, a series of ions of PTs were obtained from the LC–MSn analyses of the crude extract of A. orientalis and the ions with frequent occurrence were selected as featured ions. The peaks observed in the LC–MSn analyses were classified into different families based on the characteristic ions. The family networks were built via the diagnostic ions, which were shared in more than two families referring to the global identification method. Then the peaks detected in the LC–MSn analyses were identified selectively and sensitively through such strategies, summarized fragmentation rules and database querying. The diagnostic ions for different types of PTs were illuminated finally. Under the guide of above method, we investigated the chemical constituent of alismatis rhizoma and obtained three compounds. The summarized diagram of the approaches for researching the PTs in alismatis rhizoma was shown in Fig. 1. 2.2. Materials and samples The dried alismatis rhizoma were purchased from Sichuan province in October 2012 and identified by Prof. Jun Zhou. A voucher specimen (No. 2012-1008) was deposited with the Laboratory of Antivirus and Natural Medicinal Chemistry, Kunming Institute of Botany, Chinese Academy of Science. The reference compounds I–IV were isolated from the rhizomes of Alisma orientalis Juzep. in our laboratory, whose structures were unambiguously determined by numerous spectroscopic data [2,8]. The reference compounds I–IV were prepared by dissolving each sample in a solution of 70% CH3 CN/H2 O (v/v) to a final concentra-

tion of 0.5 mg/mL. The samples were introduced into the source via a syringe pump at a flow rate of 3 uL/min. 2.0 g dried powder of alismatis rhizoma was extracted with 20 mL acetonitrile by ultrasonication (30 min, twice). The combined CH3 CN extracts were evaporated to concentrate about 5 mL and centrifuged (10 000 rpm, 5 min, twice) before LC–MS analyses. The general chemical samples and instruments were presented in supporting information.

2.3. Liquid chromatography and mass spectrometry methods Shimadzu LCMS–IT–TOF system (Shimadzu, Kyoto, Japan) was used to analyze all the samples. The injection volume was 5 uL for LC–MS analysis. The mobile phases for LC–MS analysis were A: formic acid/water (0.05/100, v/v) and B: formic acid/acetonitrile (0.05/100, v/v) and delivered at a flow rate of 0.2 mL/min utilizing a binary gradient elution as follows: 25% B initial for origination, linear gradient 25–90% B from 0 to 30.0 min; isocratic gradient 90% B from 30.0 to 32.0 min, then returned to initial 25% B from 32.0 to 34.0 min and sustained until 36.0 min for column balance. The detailed liquid chromatography instruments and mass spectrometry methods were listed in the supporting information. MSn experiments for the compounds I–IV were achieved in manual pattern, and LC–MSn analyses for the crude extract were performed in an automatic mode for three stages MS experiments. Accurate masses were corrected by calibration using the sodium trifloroacetate (CF3 CO2 Na) clusters. The MSn analytical conditions were as follows: both positive and negative ion mode, drying gas pressure, 100.0 kPa; nebulizing gas (N2 ) flow, 0.5 L/min; spray voltage, −3.50 kV and +4.5 KV; detector voltage, 1.60 kV; equipment temperature, 40.0 ◦ C; heat block temperature, 200.0 ◦ C; curved desolvation line (CDL) temperature, 200.0 ◦ C; precursor ion selected width, m/z ±3.0 Da, and selected time, 20 ms; collision induced dissociation (CID) collision time, 30 ms; ion accumulation time, 10 ms; collision energy, 50%; collision gas argon, 50%; and q = 0.251; scan range, m/z 100–1000 for MS and MSn . The Shimadzu Composition Formula Predictor was used to speculate the molecular formula.

2.4. Extraction and LC–MS guided isolation Alismatis rhizoma (1.0 kg) was ultrasonic extracted (53 KHz) with acetone (3 L) for 3 times, 30 min for each time. The combined extract (60 g) was concentrated in vacuo and subjected on silica gel column chromatography (SiCC, 600 g, 9.5 × 30 cm) eluted with petroleum ether–acetone gradient (from 80:20 to 50:50) to give seven fractions (Frs. 1–7, 3.5 g, 11.0 g, 15.0 g, 3.0 g, 8.0 g, 10.5 g and 2.0 g). According to LC–MS analysis, Fr. 2 (11.0 g) showed the targeted peaks (peaks 15, 16 and 18) and was further separated by column chromatography (SiCC, 250 g, 5 × 30 cm) eluted with petroleum ether–chloroform (90:10) to give eight subfractions (Frs. 2-1–2-8, 900 mg, 1.2 g, 2.1 g, 800 mg, 750 mg, 1.5 g, 1.0 g and 2.0 g). Fr.2-8 (2.0 g) with the targeted peaks under the guide of LC–MS was subjected on Sephadex LH-20 CC (MeOH CHCl3 , 50:50) to give five subfractions (Frs. 2–8-1–2-8-5, 180 mg, 200 mg, 600 mg, 450 mg and 300 mg). The targeted fraction Fr. 2–8-3 (600 mg) was further subjected on silica gel column chromatography (SiCC, 300 g, 2 × 25 cm) eluted with CHCl3 –EtOAc gradient (30:1) to obtain fractions (Frs. 2–8-3-1–2-8-3-3, 100 mg, 150 mg and 250 mg). LC–MS analysis by UFLCMS-IT-TOF was performed on Fr. 2–8-3-2 to provide compounds 15, 16 and 18. Fr. 2–8-3-2 (150 mg) was subsequently purified by HPLC using MeCN H2 O (80:20) as the mobile phase to generate compound 15 (25 mg, tR = 22 min), compound 16 (7 mg, tR = 23 min) and compound 18 (20 mg, tR = 24.5 min).

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model

ARTICLE IN PRESS

MASPEC-15745; No. of Pages 10

X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

3

Fig. 1. Summary diagram of the approaches for research the PTs in alismatis rhizoma.

3. Results and discussion 3.1. MSn of the reference compounds I–IV The MSn analyses of reference compounds I–IV (Fig. 2) were performed in a manual mode in both positive and negative mode from which the [M+H−H2 O]+ ions were easily observed with high abundance. So the [M+H−H2 O]+ ions were selected as precursor ions to display MSn analyses in the positive mode. The data for accurate masses and molecular formula of the reference compounds I–IV observed from MSn were shown in Table S1. The fragmentation pathways of alisol A (I) and alisol F (IV) were shown in Fig. 2, and that of alisol A 24-acetate (II) and aliso B 23-acetate (III) were exhibited in Fig. S1. It was found that the [M+H]+ ions were hardly obtained in the MS analyses of the reference compounds I–IV except alisol B 23-acetate which gave rise to the [M+H]+ ion at m/z 515 with low intensity (40%). The [M+H−H2 O]+ ion with high abundance was attributed to the elimination of C(11)-hydroxyl group as one molecule of H2 O. The further losses of hydroxyl, acetyl and side chain groups were the main fragment pathways in the MS2 analyses of [M+H−H2 O]+ ion that generated a series of common ions at m/z 383 (381), 365 and 339. Fig. 2 showed the detailed approaches they came from. Furthermore, the intensity of the common ions was influenced by the substituent groups on the PTs. Alisol A (I) without an acetyl group on the chain side afforded ions at m/z 365 with medium abundance (67%). Alisol A 24-acetate (II) with C(24)-acetatyl group and alisol B 23-acetate (III) with C(23)-acetyl unit generated ion at m/z 365 as the base peak ion, but the intensity of ion at m/z 383 was lower than that of aliosl A (I). It suggested that the intensity of ions at m/z 383 and 365 could be regarded as the guideline to distinguish an acetyl group on the chain side or not. The ion at m/z 365 was not observed in the MSn analysis of alisol F (IV) owing to the hexatomic ring between C(16) and C(23) positions which made the C(23)-side chain not be eliminated easily as an entirety and the intensity of

ion at m/z 381was also reduced compared with that of ion at m/z 383. Although the reference compounds I–IV were not enough to all the types of PTs, the main ragmentation pathway of them took place on their side chain. These fragmentation rules could be used to recognize the structures of common ions in the LC–MSn analyses of the crude extract of alismatis rhizoma and make preparation for the identification of PTs further. 3.2. LC–MSn of the crude extract of alismatis rhizoma A total of 20 PTs were identified including one new compound and nineteen known ones in the LC–MSn analyses of the crude extract of alismatis rhizoma. The peaks were marked in the LC–MS (BPC) chromatogram (Fig. 3). The retention time, accurate mass of ions, mass error, predicted chemical formulas, detailed MS2 data and identified compound names of the peaks were listed in Table 1. 3.2.1. Common ions recognition, family classification and network establishment It was found that peaks 1–20 shared a series ions such as m/z 383 (381), 365 and 339. Their structures could be identified by the fragmentation rules summarized from the reference compounds (as shown in Fig. 2). As shown in Table 2, six ions at m/z 339, 353, 365, 381, 383 and 397 with high frequency of occurrence (more than three peaks shared) were selected as common ions to act as the basis for classifying the peaks. Except for peaks 1, 2, 6, 7, 16 and 17, the other fourteen peaks were successfully classified into six families based on the common ions. Peaks 10, 11 and 13 appeared frequently in families 1, 3 and 5, thus they were chosen as the “bridging peaks” to connect the three families. The “bridging peaks” of families 1 and 4 were peaks 15 and 18. Peaks 4 and 5 were the “bridging peaks” of families 2, 4 and 6. Through such a strategy, the networks were built as shown in Fig. 4 in which the six families were in the same network.

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model

ARTICLE IN PRESS

MASPEC-15745; No. of Pages 10

X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

4

Table 1 Characterization of the peaks in LC/ESI–MSn chromatogram of alismatis rhizoma. Peak

tR (min)

Formula

MW

MSn ions in positive modes (errora in mDa and relative abundance)

Name

1 2

8.44 9.91

C30 H48 O6 C32 H50 O7

504 547

16-oxoalisol A [19] 16-oxoalisol A 24-acetate [13]

3 4

10.54 11.65

C30 H50 O6 C30 H46 O5

506 486

5

12.40

C32 H48 O6

528

6 7

12.58 12.80

C32 O50 O6 C32 O50 O6

530 530

8 9

13.43 13.75

C32 O48 O6 C30 O44 O4

528 468

10

15.17

C30 H50 O5

490

11

15.59

C32 H52 O6

532

12

15.96

C32 H50 O6

530

13

17.10

C32 H52 O6

532

14

18.48

C30 H48 O4

472

15

19.78

C30 H46 O4

470

16

20.25

C30 H48 O4

472

17

20.71

C32 H50 O4

498

18

21.17

C32 H48 O5

512

19

21.64

C32 H50 O5

514

20

22.34

C32 H50 O5

514

Pos: [M+H]+ 505.3600 (+7.6) MS2 : 505 → 487.3386 (C30 H47 O5 , −3.2, 67%) → 469.3369 (C30 H45 O4 , +5.7, 100%) → 451.3292(C30 H43 O3 , +8.5, 38%), 505 → 415.2792 (C26 H39 O4 , −5.1, 76%) Pos: [M+H]+ 547.3695 (+6.6) MS2 : 547 → 529.3508 (C32 H49 O6 , −1.6) → 469.3285 (C30 H45 O4 ,−2.7, 29%), 529 → 451.3224 (C30 H43 O3 ,+1.7, 100%) → 433.3018 (C30 H41 O2 , −8.3, 52%) Neg: [M+HCOO]− 551.3630 (+4.1) MS2 : 507 → 489.3559 (C30 H49 O5 , −1.6, 50%) 507 → 471.3428 (C30 H47 O4 , −4.1, 100%) → 453.3362(C30 H45 O3 , −0.1, 70%) → 381.2882 (C26 H37 O2 , +9.4, 100%) → 337.2393 (C24 H33 O, −13.3, 70%) Pos: [M+H]+ 487. 3415 (−0.3) Neg: [M+HCOO]− 531.3375 (+5.2) MS2 : 487 → 469.3288 (C30 H45 O4 , −2.4, 29%) → 451.3229 (C30 H43 O3 , +2.2, 29%) 487 → 397.2716 (C26 H37 O3 , −2.1, 100%) → 353.2448 (C24 H33 O2 , −2.7) 469 → 381.2757(C26 H37 O2 , −3.8, 100%) Pos: [M+H]+ 529.3542 (+1.8) MS2 : 529 → 511.3468 (C32 H47 O5 , +5.0, 100%) → 451.3229 (C30 H43 O3 , +2.2, 58%) → 433.3102 (C30 H41 O2 , +0.1, 100%), 451 → 381.2739 (C26 H37 O2 , −4.9, 18%) 451 → 397.2793 (C26 H37 O3 , +5.6, 35%) → 353.2448 (C24 H33 O2 , −2.7, 35%) Pos:[M+H]+ 531.3734 (+5.4) MS2 : 531 → 413.2709 (C26 H37 O4 , +2.3, 100%) → 395.2593 (+1.2, C26 H35 O3 , 100%) Pos: [M+H]+ 531.3611 (−6.9) MS2 : 531 → 513.3655 (C32 H49 O5 , +8.0, 100%) → 453.3379 (C30 H45 O3 , +1.6, 30%) 513 → 435.3267 (C30 H43 O2 , +1.8, 100%), 513 → 399.2788 (C26 H39 O3 , −10.6) Pos: [M+H]+ 529.3535 (+1.1) Neg: [M+HCOO]− (573.3433, −0.3) MS2 : 529 → 511.3427 (C32 H47 O5 , +0.9, 100%) → 451.3206 (C30 H43 O3 , −0.1, 85%) → 433.3108 (C30 H41 O2 , +0.7, 100%), 451 → 397.2742 (C26 H37 O3 , +0.5, 40%) Pos: [M+H]+ 469.3314 (+0.2) MS2 : 469 → 451.3211(C30 H43 O3 , +0.4, 38%) 469 → 397.2765 (C26 H37 O3 , +2.8, 100%) → 353.2414 (C24 H33 O2 , −6.1, 25%) Neg: [M+HCOO]− 535.3658 (+1.8) MS2 : 491 → 473.3633 (C30 H49 O4 , +0.8, 100%) → 455.3522 (C30 H47 O3 , +0.2, 52%) → 437.3524 (C30 H45 O2 , +1.0, 33%), 473 → 383.2975 (C26 H39 O2 , +3.0, 100%) → 365.2895 (C26 H37 O, +5.6, 67%)→ 339.2674 (C24 H35 O, −0.8, 29%) Neg: [M+Cl]− 567.3412 (−4.6), [M+HCOO]− 577.3751 (+0.5) MS2 : 533 → 515.3719 (C32 H51 O5 , −1.2, 100%) → 497.3596 (C32 H49 O4 , 497.3625, −2.9, 100%) → 437.3339 (C30 H45 O2 , −7.9, 67%) → 419.3383 (C30 H43 O, +7.5, 33%) 497 → 383.2889 (C26 H39 O2 , −5.6, 33%) 497 → 365.2898 (C26 H37 O, +5.9, 100%) → 339.2771 (C24 H35 O, +8.9, 90%) Neg: [M+HCOO]− 575.3598 (+0.9) MS2 : 531 → 513.3539 (C32 H49 O5 , −3.6, 100%) → 339. 2654 (C24 H35 O, −2.8, 100%) 531 → 471.3532 (C30 H47 O4 , +6.3, 47%) Neg: [M+Cl]− 567.3425 (−3.3), [M+HCOO]− 577.3731 (−1.5) MS2 : 533 → 515.3725 (C32 H51 O5 , −0.6, 100%) → 497.3704 (C32 H49 O4 , +7.9, 15%) → 437.3413(C30 H45 O2 , −0.1, 45%) → 419.3316 (C30 H43 O, +0.8, 30%) 437 → 383.2942 (C26 H39 O2 , −0.3, 25%), 515 → 365.2837 (C26 H37 O, 365.2839, −0.2, 100%) → 339.2677 (C24 H35 O, 339.2682, −0.5, 50%) Pos: [M+H]+ 473.3621 (−0.4), Neg: [M+HCOO]− 517.3535 (0) MS2 : 473 → 455.3472 (C30 H47 O3 ,−4.8, 100%) → 437.3370 (C30 H45 O2 , −4.4, 67%) → 419.3381 (C30 H43 O, +7.3, 15%), 455 → 383.2948 (C26 H39 O2 , +0.3, 100%) Pos: [M+H]+ 471.3510 (+4.1), [M+Na]+ 493.3246 (−4.2) MS2 : 471 → 395.2944 (C27 H39 O2 , −0.1, 42%) → 381.2757 (C26 H37 O2 , −3.1, 42%) 471 → 339.2693 (C24 H35 O, +1.1, 100%) Neg: [M+Cl]− 551.3496 (−1.3), [M+HCOO]− 561.3754 (−4.3) MS2 : 473 → 455.3526 (C30 H47 O3 , +0.6, 100%) 473 → 383.2938 (C26 H39 O2 , −0.7, 81%) → 365.2771 (C26 H37 O, −6.8, 29%) Pos: [M+H]+ 499.3791 (+0.9) MS2 : 499 → 439.3581 (C30 H47 O2 , +1.0, 67%) → 421.3482(C30 H45 O, +1.7, 58%) → 367.2984 (C26 H39 O, −1.1, 46%) Pos: [M+H]+ 513.3629 (+5.4) MS2 : 513 → 495.3519 (C32 H47 O4 , +5.0, 65%) → 381.2826 (C26 H37 O2 , +3.8, 48%) → 339.2733 (C24 H35 O, +5.1, 40%) Pos: [M+H]+ 515.3755 (+2.4) [M+Na]+ 537.3617 (+6.7) MS2 : 515 → 497.3619 (C32 H49 O4 , −0.6, 100%) → 383.3032 (C26 H39 O2 , +8.7, 28%) → 339.2613 (C24 H35 O, −6.9, 25%) Pos: [M+H]+ 515.3707 (−2.4), [M+Na]+ 537.3617 (+6.7) MS2 : 515 → 455.3586 (C30 H47 O3 , +6.6, 60%) → 383.2956 (C26 H39 O2 , +1.1, 54%) → 365.2800 (C26 H37 O, −3.9, 35%)

a b

13␤, 17␤-epoxylalisol A [2] alisol C [19]

alisol C 23-acetate [13]

alisol N 23-acetate [20] 16␤-hydroxylalisol B 23-acetate [21]

alisol Q 23-acetate [21] alisol L [20]

alisol A [21]

alisol A 23-acetate [11] or alisol E 23-acetate [22]

alisol F 24-acetate [2]

alisol A 24-acetate [2]

25-anhydroalisol A [2]

24-deacetyl alisol O [23]

11-deoxylalisol Fb

11-deoxylalisol B 23-acetate [19] alisol O [2]

alisol B 23-acetate [2]

11-deoxyl-13␤, 17␤-epoxyalisol B 23-acetate [19]

The difference between measured and theoretical values. New compounds characterized.

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model MASPEC-15745; No. of Pages 10

ARTICLE IN PRESS X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

5

Fig. 2. The chemical structures of the reference compounds I–IV and the proposed fragmentation pathways of alisol A (I) and alisol F (IV).

3.2.2. Compounds characterization and diagnostic ions illumination The structures of identified compounds were shown in Fig. S2. Based on the established networks, the fragmentation rules and references, the structure characterization processes were described as follows in detail. 3.2.2.1. Compounds in family 2, 4 and 6, and diagnostic ions in type a and C PTs. Ions at m/z 353 (C24 H33 O2 , F2), 381 (C26 H37 O2 , F4) and 397 (C26 H37 O3 , F6) were the common ions of peaks 4 and 5 which presented the carbonyl group on the fragments compared with the structures of ions at m/z 339 (C24 H35 O), 365 (C26 H37 O) and 383 (C26 H39 O2 ) in the MSn analyses of reference compounds. Peak 4 with the molecular formula of C30 H46 O5 could be characterized as alisol C and peak 5 was alisol C 23-acetate as the monoacetate of alisol C with the chemical formula of C32 H48 O6 , both structures

Table 2 Diagnostic ions, ions determination and family classification for the peaks and skeletons. Family No.

Diagnostic ions (m/z)

Chemical formula

Peaks of each family

Types

1 2 3 4 5 6

339 353 365 381 383 397

C24 H35 O C24 H33 O2 C26 H37 O C26 H37 O2 C26 H39 O2 C26 H37 O3

10, 11, 12, 13, 15, 18,19 4, 5, 9 10, 11, 13, 20 3, 4, 5, 15, 18 10, 11, 13, 14, 19, 20 4, 5, 8, 9

B A B A/C B A

were with C(16)-carbonyl group in consideration of the results from database finding. Peaks 4 and 5 were classified into type A PTs with C(11)-hydroxyl group and type B with C(11)-anhydroxyl unit. All the peaks presented the diagnostic ion at m/z 353, 381

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model MASPEC-15745; No. of Pages 10 6

ARTICLE IN PRESS X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

Fig. 3. Base peak chromatogram (BPC) of alismatis rhizoma in both positive (1BPC) and negative ion mode (4BPC).

Fig. 4. Established networks by common ions.

and 397 which could be regarded as the diagnostic ion for type A PTs. Peak 12 was assigned to alisol F 24-acetate concurs with the fragmentation routes in which the [M+H−H2 O]+ ion at m/z 513.3539, [M+H-AcOH]+ ion at m/z 471.3532 and featured ion at m/z 339.2654 were all detected. Peaks 15 and 18 were the “bridging components” between family 1 and family 4 which possessed the characteristic ions at m/z 339 and 381. It was found that the fragmentation rules of 24-deacetyl alisol O and alisol O in references were in accordance with those of peaks 15 and 18. Consequently, peaks 15 and 18 were diagnosed as 24-deacetatetylalisol O and alisol O respectively. The common ions at m/z 381 and 339 were deemed as the diagnostic ions of peaks 15 and 18 which were classified into type C PTs. The molecular formula of peak 3 was C30 H50 O6 . Ions at m/z 489, 471 and 453 were ascribed as the [M+H−nH2 O]+ ions generating from the losses of hydroxyl units in the structure. The [M+H−3H2 O]+ ion further eliminated the C(23)-side chain group to afford the common ion at m/z 381.2882 (C26 H37 O2 , F5). It was found that 13␤, 17␤-epoxylalisol A conformed the fragmentation behaviors of peak 3 through references. Peak 3 was reasonably determined as 13␤, 17␤-epoxylalisol A. The molecular formula of peak 9 was C30 H44 O4 to exhibit the common ion at m/z 397. The product ion at m/z 451.3211 could be explained by the loss of one hydroxyl unit from the [M+H]+ ion of peak 9. Accordingly, peak 9 could be identified as alisol L. Peak 8 with the molecular formula of C32 O48 O6 gave rise to the [M+H−H2 O]+ ion at m/z 511, the [M+H−H2 O−AcOH]+ ion at m/z 451 and the common ion at m/z 397. Through references, only alisol Q 23-acetate met with the fragmentation behaviors. Alisol Q 23acetate eliminated C(17)-hydroxyl group to generate ion at m/z 511 and further afforded an ion at m/z 451 by losing C(23)-acetyl unit. Thus peak 8 was identified as alisol Q 23-acetate and the structure

of the common ion at m/z 397 was with C(11)-carbonyl rather than C(16)- carbonyl group. 3.2.2.2. Compounds in family 1, 3 and 5, and diagnostic ions in type B PTs. The structures of ions at m/z 339 (F1), 365 (F3) and 383 (F5) could be identified based on the reference compounds. Peaks 10, 11 and 13 presented all the three diagnostic ions which were similar with those of alisol A. The molecular formula and fragmentation pathways of peak 10 were the same as the reference compound alisol A (I), therefore peak 10 was determined as alisol A. Peaks 11 and 13 displayed the same molecular formula as C32 H52 O6 which was 42 Da more than alisol A. The fragmentation rule of m/z 497 → 437 generated by the elimination of one molecule of AcOH (−60 Da) was detected, which demonstrated that peaks 11 and 13 were alisol A monoacetate. The fragmentation behaviors of peak 13 were in accord with the reference compound alisol A 24-acetate (II), thus peak 13 was characterized as alisol A 24-acetate. Peak 11 might be alisol E 23-acetate or alisol A 23-acetate, a pair of optical isomers. Peak 14 gave rise to the common ion at m/z 383 and 339 just as alisol A, the structure was confirmed to be 25-anhydroalisol A by the molecular formula of C30 H48 O4 which was 18 Da less than alisol A. Peak 19 was identified as alisol B 23-acetate unambiguously due to the same fragment pathways as the reference compound III. Thus peaks 10, 11, 13, 14 and 19 were classified into type B PTs shared diagnostic ions at m/z 383, 365 and 339. Peak 20 was classified into families 3 and 5 due to the existence of product ions at m/z 365 and 383. The [M+H−AcOH]+ ion at m/z 455.3586 with high intensity suggested there was an acetyl unit on the side chain and the [M+H−H2 O]+ ion was not detected, indicating the absence of 11-hydroxyl group. Peak 20 was identified as 11-deoxyl-13␤, 17␤-epoxyalisol B 23-acetate in accordance with the further database querying. The ion at m/z 383 came from the ion at m/z 455 by the loss of all the side chain groups at C(23) position and then ion m/z 383 eliminated 13␤, 17␤-epoxyl as one

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model MASPEC-15745; No. of Pages 10

ARTICLE IN PRESS X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

7

Fig. 5. UFLCMS-PDA analysis of Fr. 2-8-3-2.

molecular of H2 O to give the ion at m/z 365. The generations of diagnostic ions at m/z 383 and 365 in the MS2 analyses of peak 20 were different from that of type B PTs. 3.2.2.3. Compounds not classified into any families. Peaks 1, 2, 6, 7, 16 and 17 were not classified into any families. The determinations of them relied on the information acquired from database querying. Peaks 1 and 2 presented similar fragmentation pathways to generate [M+H-nH2 O]+ ions at m/z 529, 487, 451 and 433, indicating the presence of PTs with polyhydroxyl substituent groups. Peak 1 could be determined as 16-oxoalisol A in line with the outcome in database. It was speculated that peak 2 might be the monoacetate of peak 1 according with the molecular formulas of C32 H50 O7 and

peak 2 was illuminated as 16-oxoalisol A 24-acetate. Thus peaks 1 and 2 were classified into type A PTs with C(16)-carbonyl unit. Both the molecular formulas of peaks 6 and 7 were C32 O50 O6 which exhibited the [M+H]+ ion at m/z 531. Peak 6 eliminated the side chain at C(23) position directly to produce the ion at m/z 413 and further lost one molecule of H2 O to generate the ion at m/z 395. Peak 7 present the [M+H−H2 O]+ ion at m/z 513 as the base peak ion and the [M+H−H2 O −AcOH]+ ion at m/z 453 was also detected. The ion at m/z 399 (C26 H39 O3 ) of peak 7 was ascribed as the hydroxylation fragment of ion at m/z 397 and peak 7 could be regarded as the hydroxylation product of alisol C 23-acetate which named 16␤-hydroxylalisol B 23-acetate. Peak 6 was illuminated as alisol N 23-acetate with C(11), C(12)-dihydroxyl.

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model

ARTICLE IN PRESS

MASPEC-15745; No. of Pages 10

X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

8 Table 3 1 H and 13 C No.

NMR data of compounds 15, 16 and 18 (ı in ppm, J in Hz) in CDCl3 .a 24-deacetyl alisol O (15) 1

1

11-deoxylalisol F (16) 13

H NMR

C NMR

31.0 (t)

1

8 9 10 11

2.01–2.05 (1H, m) 1.60–1.65 (1H, m) 2.66–2.67 (1H, m) 2.29–2.31(1H, m) – – 2.25–2.27(1H, m) 1.45–1.50 (1H, m) 1.22–1.30 (1H, overlapped) 1.85–1.90 (1H, m) 1.22–1.30 (1H, overlapped) – 2.30 (1H, br d, 3.3) – 6.24 (1H, dd, 10.1, 2.7)

12

5.65 (1H, br d, 10.0)

129.9 (d)

13 14 15

– – 2.16 (2H, br s)

138.7 (s) 54.9 (s) 37.1 (t)

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

4.56 (1H, d, 8.5) – 0.85 (3H, s) 0.89(3H, s) 2.98 (1H, m) 1.19 (3H, d, 7.1) 1.61–1.63 (2H, m) 4.10 (1H, d, 11.9) 3.02 (1H, br s) – 1.29 (3H, s) 1.23 (3H, s) 1.04 (3H, s) 1.03 (3H, s) 1.06 (3H, s)

80.3 (d) 134.8 (s) 22.6 (q) 24.7 (q) 27.0 (d) 17.7 (q) 35.8 (t) 73.0 (d) 77.0 (d) 73.4 (s) 26.5 (q) 27.2 (q) 29.3 (q) 19.2 (q) 24.7 (q)

1.99–2.03 (1H, m) 1.38–1.47 (1H, m) 2.66–2.60 (1H, m) 2.25–2.28 (1H, m) – – 1.98–2.04 (1H, m) 1.41–1.45 (1H, m) 1.22–1.27 (1H, m) 1.98–2.04 (1H, m) 1.23–1.27 (1H, m) – 1.66 (1H, br d, 2.8) – 1.85–1.95 (1H, m) 2.29–2.33 (1H, m) 1.85–1.95 (1H, m) 2.29–2.33 (1H, m) – – 1.19–1.23 (1H, m) 2.22–2.30 (1H, m) 4.49 (1H, t) – 0.81 (3H, s) 0.89 (3H, s) 2.83 (1H, m) 1.16 (3H, d, 7.2) 1.66 (2H, dd, 12.7, 3.0) 4.03 (1H, d, 11.9) 3.05 (1H, br s) – 1.30 (3H, s) 1.24 (3H, s) 1.06 (3H, s) 1.03 (3H, s) 1.06 (3H, s)

–

– –

2 3 4 5 6 7

– a 1

H NMR recorded in 600 MHz,

13

33.5 (t) 220.0 (s) 47.1 (s) 46.2 (d) 19.2 (t) 32.1 (t) 37.9 (s) 47.1 (d) 35.8 (s) 120.9 (d)

alisol O (18) 13

H NMR

C NMR

31.6 (t)

1

H NMR

13

C NMR

31.0 (t)

40.6 (s) 43.7 (d) 36.2 (s) 22.9 (t)

2.02–2.04 (1H, m) 1.69–1.70 (1H, m) 2.68–2.76 (1H,m), 2.29–2.32 (1H, m) – – 2.29–2.32 (1H, m) 1.48–1.50 (1H, m) 1.22–1.30 (1H, m) 1.85–1.93 (1H, m) 1.22–1.30 (1H, m) – 2.30 (1H, br d, 3.3) – 6.25 (1H, dd, 10.1, 3.0)

22.3 (t)

5.68 (1H, br d, 10.1)

130.2 (d)

139.1 (s) 55.5 (s) 39.9 (t)

– – 2.17 (2H, br s)

139.1 (s) 55.0 (s) 36.9 (t)

80.0 (d) 131.5 (s) 22.6 (q) 23.5 (q) 26.5 (d) 18.4 (q) 34.8 (t) 72.6 (d) 77.2 (d) 73.4 (s) 26.5 (q) 27.0 (q) 29.3 (q) 19.6 (q) 24.4 (q)

4.58 (1H, d, 8.5) – 0.85 (3H, s) 0.90 (3H, s) 2.97 (1H, m) 1.19 (3H, d, 7.2) 1.67 (2H, ddd, 12.7, 11.9, 5.3) 4.32 (1H, d, 12.4) 4.70 (1H, d, 1.6) – 1.36 (3H, s) 1.12 (3H, s) 1.07 (3H, s) 1.04 (3H, s) 1.08 (3H, s)

81.0 (d) 134.1 (s) 22.6 (q) 24.6 (q) 27.2 (d) 17.6 (q) 35.8 (t) 72.8 (d) 77.2 (d) 72.8 (s) 26.6 (q) 27.9 (q) 29.3 (q) 19.2 (q) 24.7 (q)

–

–

–

171.2 (s)

–

–

2.17 (3H, s)

20.8 (q)

33.6 (t) 220.2(s) 47.0 (s) 47.8 (d) 19.9 (t) 33.6 (t)

33.5 (t) 220.0 (s) 47.1 (s) 46.2 (d) 19.2 (t) 32.2 (t) 38.0 (s) 47.1 (d) 35.8 (s) 120.9 (d)

C NMR recorded in 150 MHz.

Peak 16 presented the [M+Cl]+ ion at m/z 551.3496 and [M+HCOO]+ ion at m/z 561.3754, proposing its molecular formula was C30 H48 O4 . It might be the product of alisol F without C(11)hydroxyl group and peak 16 was determined as 11-deoxylalisol F as a new compound. Peak 17 with the molecular formula of C32 H50 O4 showed the product ions at m/z 367 (C26 H39 O) and 421 (C30 H45 O), due to the loss of side chain and was ascribed as 11-deoxylalisol B 23-acetate which was the only object matched the fragmentation rules in database querying. 3.3. Characterization of compound 15, 16 and 18 (1 H NMR and 13 C NMR data see Table 3) LC–MS analysis by UFLCMS-IT-TOF was performed on Fr. 2-8-32 (Fig. 5). Compound 15, 16 and 18 were obtained from the targeted fraction under the guide of such method . Compound 16 was isolated as a white crystal and had a molecular formula of C30 H48 O4 by HRESIMS at m/z 495.3434 [M+Na]+ (calcd 495.3445) in the positive mode and m/z 561.3754 [M+HCOO]− (calcd 561.3797) in the negative mode with 7 ◦ of unsaturation. The fragmentation pathways, 473 → 455.3526 (C30 H47 O3 , +0.6, 100%) and 473 → 383.2938 (C26 H39 O2 , −0.7, 81%) → 365.2771 (C26 H37 O, −6.8, 29%) indicated that it was PTs

as the deoxyl product of aliosl F in above analysis. In accordance with its molecular formula, all the 30 carbons were well resolved in the 13 C NMR (DEPT) spectrum to be eight methyl, six methylenes, eight methines and eight quaternary carbons. Compared with the 13 C NMR spectrum of 24-deacetyl alisol O [20], compound 16 lost a methylene signal at the range of ıC 70–80 owing to the C(11) of alisol F. Furthermore, methylene signals at ıC 120 and 129 were not find in the NMR spectrum of compound 16 compared to those of 24-deacetyl alisol O (ıC 120, C-11and ıC 129, C-12) [23], which suggested that compound 16 might be the C(11)-deoxyl product of aliosl F, too (Fig. 6). The 1 H–1 H COSY correlations of ıH (1H, 1.66, d, J = 2.8 Hz, H-9)/ıH (2H, 2.29–2.33, 1.85–1.95, H-12)/ıH (2H, 2.29–2.33, 1.85–1.95, H-11) and HMBC correlations from ıH (1H, 1.66, d, J = 2.8 Hz, H-9) to ıC (22.9, C-11) confirmed that a hydroxyl group was absent in C(11) position. All the above evidence proved compound 16 was the C(11)-deoxyl product of aliosl F and named as 11-deoxylalisol F. Compound 16 was a new compound with an overall new structure which is discovered in alismatis rhizoma the first time. 24-Deacetyl alisol O, compound 15, white acicular crystal, the molecular formula was C30 H46 O4 by HRESIMS at m/z 471.3510 [M+H]+ (calcd 471.3551) and 493.3246 [M+Na]+ (calcd 493.3204) in the positive mode. 1 H and 13 C NMR data were provided in Table 3 [23].

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model MASPEC-15745; No. of Pages 10

ARTICLE IN PRESS X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

9

Fig. 6. (A) the13 C NMR spectrum of compound 15; (B) the 13 C NMR spectrum of compound 16; (C) the magnified 13 C NMR spectrum of compound 16; (D) the key 2D-NMR correlations of compound 16.

Alisol O, compound 18, white acicular crystal, the molecular formula was C32 H48 O5 by HRESIMS at m/z 513.3629 [M+H]+ (calcd 513.3575) in the positive mode. 1 H and 13 C NMR data were listed in Table 3 [2]. 4. Conclusion 20 PTs including 1 new compound and 19 known ones were identified by LCMS-IT-TOF combined with the global identification strategy. The most of the determined compounds were classified into type A–C PTs according to the difference in structures and their diagnostic ions were also illuminated: ions at m/z 353, 381 and 397 for type A, ions at m/z 339, 365 and 383 for type B and ion at m/z 381 for type C. The diagnostic ions were the guidelines for analyzing PTs in alismatis rhizoma by mass spectrometry. The new compound 11-deoxylalisol F (16) accompanying with two known compounds 24-deacetattetylalisol O (15) and alisol O (18) were obtained from alismatis rhizoma under the guidance of such

strategy. In conclusion, we employed four reference compounds for tandem mass analysis to summarize the fragmentation pathways which played an important role in the establishment of the global identification strategy. At the same time, the characterized new compound was obtained and determined by NMR spectrum. This study used in alismatis rhizoma was an example to demonstrate the powerful analytical ability of LCMS −IT-TOF combined with the global identification strategy for analyzing and separating PTs or other natural products.

Acknowledgements This work was supported by the Youth Innovation Promotion Association, Chinese Academy of Sciences, the Hundred Talents Program of the Chinese Academy of Sciences, the Program of Yunling Scholar, and the West Light Foundation of the Chinese Academy of Sciences (Western Youth Scholars “A”).

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004

G Model MASPEC-15745; No. of Pages 10 10

ARTICLE IN PRESS X. Chen et al. / International Journal of Mass Spectrometry xxx (2017) xxx–xxx

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijms.2017.01.004. References [1] T. Tian, H. Chen, Y.Y. Zhao, Traditional uses, phytochemistry, pharmacology, toxicology and quality control of Alisma orientale (Sam.) Juzep: a review, J. Ethnopharmacol. 158 (2014) 373–387. [2] Z.Y. Jiang, X.M. Zhang, F.X. Zhang, N. Liu, F. Zhao, J. Zhou, J.J. Chen, A new tritenpene and anti-hepatitis B virus active compounds from Alisma orientalis, Planta Med. 72 (2006) 951–954. [3] L.X. Wang, Q.N. Wu, Q. Zhang, G.P. Peng, A.W. Ding, Basic study of diuretic active compounds in rhizoma alismatis, West China J. Pharm. Sci. 23 (2008) 670–672. [4] J.G. Qin, Y.H. Wang, J.P. Liang, Y. Zhang, G.R. Wang, W.Q. Guo, Effect of alismaceae on regulating the expression of liver basal membrane heparan sulfate proteoglycan in ApoE-knock out atherosclerosis mice, Chin. Arch. Trad. Chin. Med. 25 (2007) 696–698. [5] H.R. Lin, Triterpenes from Alisma orientalis act as farnesoid X receptor agonists, Bioorg. Med. Chem. Lett. 22 (2012) 4787–4792. [6] S.M. Lee, Y.H. Kho, B.S. Min, J.H. Kim, M.K. Na, S.J. Kang, H.Y. Maeng, K.H. Bae, Cytotoxic triterpenoides from alismatis rhizome, Arch. Pharm. Res. 24 (2001) 524–526. [7] J.H. Lee, Y.J. Lee, S.W. Kang, Y. Kim, M. Shin, M. Hong, E.K. Seo, S.H. Kim, S.Y. Nah, H. Bae, Effects of protostane-type triterpenoids on the 5-HT3 A receptor-mediated ion current in Xenopus oocyte, Brain Res. 1331 (2010) 20–27. [8] Q. Zhang, Z.Y. Jiang, J. Luo, P. Cheng, Y.B. Ma, X.M. Zhang, F.X. Zhang, J. Zhou, J.J. Chen, Anti-HBV agents. Part 1: synthesis of alisol A derivatives: a new class of hepatitis B virus inhibitors, Bioorg. Med. Chem. Lett. 18 (2008) 4647–4650. [9] Q. Zhang, Z.Y. Jiang, J. Luo, J.F. Liu, Y.B. Ma, R.H. Guo, X.M. Zhang, J. Zhou, J.J. Chen, Anti-HBV agents. Part 2: synthesis and in vitro anti-hepatitis B virus activities of alisol A derivatives, Bioorg. Med. Chem. Lett. 19 (2009) 2148–2153. [10] Q. Zhang, Z.Y. Jiang, J. Luo, Y.B. Ma, J.F. Liu, R.H. Guo, X.M. Zhang, J. Zhou, W. Niu, F.F. Du, L. Li, C. Li, J.J. Chen, Part 3: preliminary structure–activity relationships of tetra-acylalisol A derivatives as potent hepatitis B virus inhibitors, Bioorg. Med. Chem. Lett. 19 (2009) 6659–6665. [11] Y. Li, X. Pan, J.P. Xiao, Y. Chen, J.Z. Chen, Determination of alisol a 24-acetate and alisol B 23-acetate in wulingsan by HPLC-ELSD, Chin. Pharm. J. 42 (2007) 1499–1501.

[12] X.F. Zhao, L.Q. Kong, Compound analysis of Alisma orientalis (Sam.) Juzep. by HPLC-EMI-MS, Chin. Trad. Planta Med. 29 (2007) 1805–1807. [13] X. Liu, S.L. Li, Y. Zhou, J.Z. Song, Y.F. Zheng, G.P. Peng, H.X. Xu, Characterization of protostane triterpenoids in Alisma orientalis by ultra- performance liquid chromatography coupled with quadrupole time- of- flight mass spectrometry, Rapid. Commun. Mass Spectrom. 24 (2010) 1514–1522. [14] C.A. Geng, H. Chen, X.L. Chen, X.M. Zhang, L.G. Lei, J.J. Chen, Rapid characterization of chemical constituents in Saniculiphyllum guangxiense by ultra fast liquid chromatography with diode arraydetection and electrospray ionization tandem mass spectrometry, Int. J. Mass Spectrom. 361 (2014) 9–22. [15] X.L. Chen, C.A. Geng, J.J. Chen, A fragmentation study on four C19-diterpenoid alkaloids by electrospray ionization ion-trap time-of-flight tandem mass spectrometry, J. Asian Nat. Prod. Res. 17 (2015) 915–929. [16] X.L. Chen, C.A. Geng, J.J. Chen, A fragmentation study of six C21 steroidal aglycones by electrospray ionization ion-trap time-of-flight tandem mass spectrometry, Nat. Prod. Commun. 10 (2015) 2053–2058. [17] B. Avula, Y.H. Wang, R.S. Pawar, Y.J. Shukla, T.J. Smillie, I.A. Khan, Identification and structural characterization of steroidal glycosides in Hoodia gordonii by ion-trap tandem mass spectrometry and liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry, Rapid Commun. Mass Spectrom. 22 (2008) 2587–2596. [18] H.P. Hao, N. Cui, G.J. Wang, B.R. Xiang, Y. Liang, X.Y. Xu, H. Zhang, J. Yang, C.N. Zheng, L. Wu, P. Gong, W. Wang, Global detection and identification of nontarget components from herbal preparations by liquid chromatography hybrid Ion trap time-of-flight mass spectrometry and a strategy, Anal. Chem. 80 (2008) 8187–8194. [19] Y. Nakajima, Y. Satoh, M. Katsumata, K. Tsujiyama, Y. Ida, J. Shoji, Terpenoids of Alisma orientale rhizome and the crude drug alismatis rhizoma, Phytochemistry 36 (1994) 119–127. [20] Y. Masayuki, T. Norimichi, M. Toshiyuki, I. Akira, M. Hisashi, M. Hideaki, K. Michinori, Studies on alismatis rhizoma. III. Stereostructures of new protostane- type triterpenes, alisols H, I, J- 23- acetate, K- 23- acetate, L- 23- acetate, M- 23- acetate, and N- 23- acetate, from the dried rhizome of Alisma orientale, Chem. Pharm. Bull. 47 (1999) 524–528. [21] H.G. Jin, Q.L. Jin, A.R. Kim, H. Choi, J.H. Lee, Y.S. Kim, D.G. Lee, E.R. Woo, A new triterpenoid from Alisma orientale and their antibacterial effect, Arch. Pharm. Res. 35 (2012) 1919–1926. [22] M. Yoshikawa, S. Hatakeyama, N. Tanaka, Y. Fukuda, J. Yamahara, N. Murakami, Crude drugs from aquatic plants. I. On the constituents of alismatis rhizoma. (1). Absolute stereostructures of alisols E 23-acetate F, and G, three new protostane-type triterpenes from Chinese alismatis rhizome, Chem. Pharm. Bull. 41 (1993) 1948–1954. [23] A.C. Zhou, C.F. Zhang, M. Zhang, A new protostane triterpenoid from the rhizome of Alisma orientale, Chin. J. Nat. Med. 6 (2008) 109–111.

Please cite this article in press as: X. Chen, et al., Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis, Int. J. Mass Spectrom. (2017), http://dx.doi.org/10.1016/j.ijms.2017.01.004