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Neutral Loss Ion Mapping Experiment Combined with Precursor Mass List and Dynamic Exclusion for Screening Unstable Malonyl Glucoside Conjugates

  • Min Yang
  • Zhe Zhou
  • Shuai Yao
  • Shangrong Li
  • Wenzhi Yang
  • Baohong Jiang
  • Xuan Liu
  • Wanying Wu
  • Hua Qv
  • De-an GuoEmail author
Research Article

Abstract

Malonates are one type of the acylation conjugates and found abundantly in ginseng and soybean. Malonyl conjugates of ginsenosides and isoflavone glycosides were often considered as the characteristic components to evaluate various species and different forms of ginseng and soybean products because of their thermal instability. Another famous isoflavonoid-rich leguminous traditional Chinese medicine (TCM), named Puerarin lobata (Gegen), has also been reported to contain malonyl daidzin and malonyl genistin. However, the conjugates were found to present in very low amount and particularly unstable in the negative ion mode scan using LTQ Orbitrap mass spectrometry with electrospray ionization (ESI). In order to screen and characterize the malonyl conjugates in Gegen, a specific method was designed and developed combining neutral loss ion mapping (NLIM) experiment and precursor mass list (PL) triggered data dependent acquisition (DDA). Along with the activation of dynamic exclusion (DE), the method was proven to be specific and efficient for searching the malonate derivatives from Gegen. Two samples were examined by the established method. A total of 66 compounds were found, and 43 of them were malonates of isoflavone glycoside. Very few compounds were reported previously in Gegen. The results are helpful to understand the constituents of Gegen with more insight. The study not only provided a method for analyzing the malonyl conjugates from complex matrices but also explored a way to trace other low amount components in TCMs.

Graphical Abstract

Key words

Malonate Ion mapping experiment Precursor mass list Dynamic exclusion Puerarin lobata Orbitrap High resolution multi-stage mass spectrometry 

Introduction

Acylglucosides are ubiquitous in higher plants because of the action of acyl-coenzyme A (CoA) transferase enzymes during the biosynthetic steps. The acylation reaction was proposed for the stabilization of labile structures, the enhancement of the solubility of target compounds in water, and the transport of target compounds into the vacuole [1]. Malonates are one type of the acylation conjugates and found abundantly in many plants [2, 3, 4, 5, 6, 7]. Ginseng and soybean are the most popular concerned species [8, 9, 10, 11, 12, 13] because of their high level of medicinal and nutritive value. Malonates are derived from a dicarboxylic acid, named malonic acid, catalyzed by the malonyl-CoA, which was considered to play an important role in the synthesis and elongation of fatty acids [14]. Despite the interests in the physiologic effect, malonyl conjugates of ginsenosides and isoflavonoid glycosides were often considered as the characteristic components to evaluate various species and different forms of ginseng and soybean products [11, 15, 16, 17] because of their thermal instability.

Puerarin lobata (Gegen) is another isoflavonoid-rich leguminous plant and has been commonly used as antipyretic and spasmolytic agent in traditional Chinese medicine (TCM) [18]. Different from soybean, Gegen contains predominantly C-glycosides besides O-glycosides. Some minor malonyl glycosides, such as malonyl daidzin and malonyl genistin, were also reported from Gegen in the past decades [19, 20, 21]. To our knowledge, there were no C-glycoside malonyl conjugates reported till now. However, in our phytochemical analysis on Gegen using ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) method in negative ion mode, no any obvious malonyl conjugates of daidzin and genistin were found. Instead, some peaks giving predominant ions at m/z 253 along with minor ions at m/z 457 and 1003 were found in full scan mass spectrum with low amount (Supplementary Figure 1S in Supporting Information). Further study was performed in positive ion mode to clarify the ambiguous components. Corresponding peaks were found to show predominant ions at m/z 503, which indicated the existence of a series of analogues of malonyl daidzin (Supplementary Figure 1S). The results showed that the malonylates were very labile in negative ion mode using electrospray ionization (ESI) and lost both malonyl and glycosyl groups in full scan mass spectrum, which did not occur on other acylates. Therefore, for fast exploration and characterization of the low amount or even trace amount malonyl glucosides in Gegen, establishment of a specific method was put on the schedule in our study.

In LC-MS analysis, the data-dependent acquisition (DDA) is a commonly used method to achieve multistage mass spectra (MSn n ≥ 3) because it can reduce the number of runs in component identification by fulfilling both survey scan and dependent scan (product ion scans) in the same run, but the success still depends on whether the correct precursor ion can trigger the dependent scan. Therefore, for rapid and comprehensive screening the target compounds, a neutral loss ion map (NLIM) experiment was considered to seek the possible precursor ions. The mapping experiment maps all precursors that produce the specified neutral loss mass in a specified mass range. In our previous study, malonyl conjugates were found to give characteristic neutral losses at m/z 86 (malonyl) or 248 (malonyl + glycosyl). Considering the presence of xylosyl and apiosyl, 218 (malonyl + xyl/api) were also selected. Hence, the NLIM experiment could be conducted to find all precursors that lost 80, 218, or 248 masses. However, some trace amounts of malonylates were covered by the large amount of uninterested ions and could not successfully trigger a dependent scan. Therefore, a precursor mass list (PL), which was obtained from the NLIM experiment, was added to the acquisition criteria to specifically trigger the malonyl conjugate precursor ions. Furthermore, dynamic exclusion (DE) was employed to resolve the completely overlapped target components by temporarily putting a mass into an exclusion list after its MSn spectrum was acquired.

Methods and Materials

Chemical and Reagents

Acetonitrile and methanol (HPLC-MS grade) were purchased from Merck KGaA (Darmstadt, Germany). Formic acid (HPLC grade) was purchased from Fisher Scientific Co. (Loughborough, UK). Deionized water (18.2 MΩ) was produced using a Milli-Q system (Millipore, Billerica, MA, USA).

3′-Hydroxypuerarin, puerarin, daidzin, calycosin-7-glucoside, genistin, ononin, daidzein, calycosin, genistein, formononetin, and biochanin A were purchased from Shanghai U-sea Biotech Co., Ltd. 3′-Methoxypuerarin, mirificin, and 3′-methoxymirificin were isolated from Radix puerariae by the authors, and the structures were identified by comparing their 1H, 13C NMR and HRMS data with the literature [22, 23].

Radix puerariae reference material was purchased from National Institutes for Food and Drug Control. Another Radix puerariae raw material was purchased from a drug store of Anhui province, China.

Sample Preparation

Powdered samples (40 mesh, 0.8 g) were accurately weighed and extracted with 10 mL methanol for 20 min with ultrasonic extraction. The solution obtained was centrifuged, and the supernatant was evaporated to dryness, and the residue was dissolved with 1 mL of methanol. One μL of the filtrate was injected into the UHPLC/HRMS system for analysis.

Chromatography Conditions

Chromatographic separation was performed using an UltiMate 3000 Binary RSLC system (ThermoFisher Scientific Inc., San Jose, CA, USA), equipped with a binary solvent delivery system, an autosampler, and a diode-array detector (DAD). An Agilent Zorbax Eclipse Plus C18 column (100 × 2.1 mm, 1.8 μm) was used for the analysis. The column temperature was set at 35°C. The flow rate was 0.5 mL/min. The mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The eluting condition was optimized as follows: holding at 9% B (0–9.5 min), then linear gradient from 9% to 16.5% B (9.5–25.5 min), from 16.5% to 23% B (25.5–33 min), from 23% to 54% B (33–44 min), then elute with 100% B for 5 min and equilibrate at 9% B for 10 min.

Mass Spectrometry

The MS instrument used was a LTQ Orbitrap Velos Pro. MS system (ThermoFisher Scientific Inc., San Jose, CA, USA). Samples were analyzed using the new high sensitive heat electrospray ionization (HESI) probe in both positive and negative ion modes, and the MS acquisition was performed from 450 to 700 Da for NLIM experiment and from 120 to 1200 Da for LC-MS acquisition at a resolving power of 60,000 (at m/z 400,FWHM). The MS source heat temperature was set at 350°C, and the capillary temperature was set at 320°C. The source voltage was 3.2 kV in negative ion mode and 3.5 kV in positive ion mode. The collision energy was set as 40% in negative ion mode and 25% (MS/MS), 35% (MS3–4) in positive ion mode. The sheath gas flow was set at 45 arb. and auxiliary gas flow was set at 5 arb.

The ion mapping experiment was performed by direct infusion at 3 μL/min in positive ion mode. The precursor mass step changed by 1.0 Da and isolation width was 2.0 Da. Collision induced dissociation (CID) was used with the normalized collision energy of 25%. The neutral loss masses were set at 86, 218, and 248 Da, respectively. The source parameters were set as: heat temperature, 60°C; capillary temperature, 320°C; source voltage, 3.5 kV; sheath gas flow, 6 arb.; auxiliary gas flow, 1 arb. The DDA was triggered by the most abundant ion from the precursor mass list, which was obtained from the NLIM experiment, followed by MSn analysis on the most abundant product ion. Dynamic exclusion was enabled when the DDA analyses was triggered with exclusion mass width at ±1.5, repeat count at 2, repeat duration at 10.0 s, exclusion list size at 50, exclusion duration at 10 s. The instrument was controlled by Xcalibur 2.2 (ThermoFisher Scientific Inc., San Jose, CA, USA).

Results and Discussion

Collision Energy Optimization Experiment for the Three Different Neutral Loss Masses

Optimization experiment was performed to obtain the optimal collision energy of the three different neutral loss masses. The collision energy varied from 10% to 35%. The neutral loss extracted ion chromatograms (XICs) for the three neutral loss masses were used to evaluate the optimal collision energy. The most abundant peaks for each neutral loss were selected as the target compound. The relationships between the abundance and different collision energy are shown in Figure 1. The abundance plateaus covered energy of 20%–25% for 86, 20%–30% for 218, and 20%–30% for 248. Noticeably, 25% was always in the optimal ranges for all of the masses. Therefore, 25% was selected as the MS/MS collision energy for both ion mapping and DDA experiments throughout the investigation.
Figure 1

Collision energy optimization for the three different neutral loss masses

Ion Mapping Experiment for Detecting the Possible Precursor Masses

The mapping experiment was based on detection and display of all ions that had lost a mass equal to the Product Ion Mass + Neutral Loss Mass ± 0.5. According to the masses of malonyl, glucosyl, and xylosyl/apiosyl, 86, 218 (86 + 132), and 248 (86 + 162) were used as target neutral losses in the NLIM experiment. The linear formula constructed a two dimension ion map in defined mass range. Adding the intensity of the response, a three dimension map was obtained (Figure 2 upper). Inputting the specified neutral loss masses in the NL mass box (Figure 2 lower), a corresponding precursor ion spectrum was mapped in the spectrum display window. The mass range was determined by the molecular masses of the isoflavone glycosides characterized from Gegen, which gave the minimum precursor ion at m/z 417 (daidzin, puerarin). Preliminary experiment showed that no obvious precursor ions were found over m/z 700. Therefore, the NLIM scan ranged from m/z 450 to 700. Each scan was completed within 4 min. The experiment resulted in a series of precursor ions at m/z 503, 513, 517, 519, 525, 531, 533, 541, 543, 547, 579, 615, and 665.
Figure 2

Exemplary view of Ion Map for experiment of neutral loss of 86 mass

DDA Analyses with Precursor Ion List and Dynamic Exclusion to Trigger the Target Precursor Ions as Much as Possible

As mentioned above, DDA provide a convenient tool to achieve MSn in a single run. Various auxiliary functions, such as precursor mass list, product mass list, neutral loss list, and dynamic exclusion, could be applied for logical selection of specific precursor ions, especially for those covered by a large amount of complex matrices. In the present study, the possible precursor ions containing malonyl were obtained by the NLIM experiment and applied in the PL, and the most intense ion of which was selected to trigger the MS/MS spectrum. DE was enabled in case the overlapped ions were both in the PL.

Figure 3 shows the neutral loss XICs of two Gegen samples. Mass of 86 originating from malonyl group was input to the Neutral Fragment box. Despite the different contents of the constituents between the two samples, the application of PL combined DE obviously improved the specificity and sensitivity of the method by triggering more precursor ions. Detailed comparison results are shown in Table 1.
Figure 3

Neutral loss XICs of two Gegen samples using 86 mass with (a), (c), or without (b), (d) PL and DE

Table 1

Results Comparison between the Designed and Normal Methods

No.

m/z

t R

YG1

YR

No.

m/z

t R

YG1

YR

N

PL + DE

N

PL + DE

N

PL + DE

N

PL + DE

4*

503.1190

12.45

 

 

28*

533.1296

22.10

 

 

17*

17.90

 

31

22.91

 

  

23*

20.11

32

23.26

 

 

26*

21.37

 

 

39*

25.94

36*

24.25

46*

27.36

 

 

42*

26.45

 

47*

27.78

 

 

27

517.1348

21.94

 

 

48*

28.03

 

 

45

27.21

 

 

53*

31.47

 

 

50*

29.78

 

 

59*

33.52

   

51

30.15

   

60*

33.78

   

58*

33.27

 

66*

37.55

62*

34.97

29

547.1448

22.33

 

 

64*

35.56

 

35

24.11

 

 

2*

519.1138

6.23

 

 

38

25.29

6*

12.22

 

 

41

26.25

 

11*

14.57

   

43

26.61

 

  

22*

19.58

 

52*

31.40

 

 

37*

24.55

 

 

54*

32.04

 

 

44*

26.76

 

 

56

32.39

 

 

49*

29.59

57

32.59

 

 

20

531.1122

19.40

 

  

63*

35.28

 

 

34

23.71

 

 

65*

36.29

 

55

32.12

 

 

1*

665.1697

5.68

 

 

61*

34.16

 

 

3*

10.92

 

  

7

533.1296

12.56

 

 

5*

11.51

 

10

14.41

 

8

12.86

 

  

13*

15.12

 

 

9*

13.96

 

  

16

17.59

 

 

12*

14.74

 

18

17.98

   

14*

15.86

 

 

19

18.37

 

 

15*

16.20

 

 

21*

19.49

30*

22.69

 

 

24

20.83

 

33*

23.49

 

 

25

21.22

 

40*

 

25.96

 

 

* Malonyl conjugates.

√ = MS/MS spectra were triggered.

YG1 = Gegen sample 1, YR = reference drug of Gegen, N = normal method, PL + DE = combined use of PL and DE.

For fully screening the target compounds, the XICs (Figure 4) of each ion in the PL were examined. The ions at m/z 503.1190, 517.1348, 519.1138, 531.1122, 533.1296, 547.1448, and 665.1697 were found to be real positive signals with accurate mass. Other precursor ions in the PL did not give obvious evidence to have malonyl group. A total of 66 compounds were characterized (Supplementary Table 1S), including a few isomers that did not contain malonyl group. Generally, most of the malonylates were derivatives of the major glycosides reported in Gegen.
Figure 4

TIC and exact full mass XICs of 503.1190, 517.1348, 519.1138, 531.1122, 533.1296, 547.1448, and 665.1697

Basic Fragmentation Patterns of Some Major Isoflavonoids in Gegen

In Gegen, most of the isoflavonoids were derivatives of 7,4′-dihydroxylisoflavone (daidzein), 5,7,4′-trihydroxylisoflavone (genistein), and 7,3′,4′-trihdyroxylisoflavone. Typical structures and MSn spectra in positive ion mode are shown in Supplementary Figures 2S–4S. For aglycons and O-glycosides, sequential losses of CO were common fragmentation pathways. Additionally, loss of H2O was observed more abundant in 7,3′,4′-trihdyroxylisoflavone than in 7,4′-dihydroxylisoflavone and 5,7,4′-trihydroxylisoflavone. Furthermore, retro-Diels-Alder (RDA) reaction produced predominant fragment ions at m/z 137.0233 for 7,3′,4′-trihdyroxylisoflavone and m/z 153.0182 for 5,7,4′-trihydroxylisoflavone, which could be used to distinguish the different substitute positions at ring A or ring B. For C-glycosides, double losses of H2O and CH2O constituted the main fragmentation patterns, which occurred in the glucosyl parts. Low abundant ions at m/z 297.0757, 313.0707, and 327.0863 presented nuclear skeleton of puerarin, 3′-hydroxypuerarin, and 3′-methoxypuerarin, respectively. Because of the lack of standard for genistin C-glycoside, the characteristic fragmentation patterns of 5-substitution isoflavone C-glycoside could only be explored from the unknown components possibly existing in Gegen.

Elaboration of the Compounds Giving Precursor Ions at m/z 503.1190

Apparently, the compounds with [M + H]+ ions at m/z 503.1190 were possibly the malonylates of puerarin or daidzin, which showed [M + H]+ ions at m/z 417.1180. In the accurate mass XIC, six peaks were extracted (Figure 4, Table 1) and all of them were triggered by the designed strategy. However, only three of them could produce MSn spectra by the normal DDA method for each sample (Table 1). Two of them (4, t R = 12.45 min; 17, t R  = 17.90 min) showed identical fragments (m/z 321.0760, 297.0761, 279.0656) and similar fragmentation patterns to those of puerarin. Hence, compounds 4 and 17 were tentatively identified as 4′(6″)-O-malonyl-puerarin.

Four other peaks (23, t R = 20.11 min; 26, t R = 21.37 min; 36, t R = 24.25 min; 42, t R = 26.45 min) gave almost identical MSn (n = 2–4) spectra. The [M + H]+ ion lost both malonyl and glycosyl and produced an ion of aglucon (daidzein). Therefore, the four compounds were identified as O-malonyl-daidzin.

Elaboration of the Compounds Giving Precursor Ions at m/z 517.1348

Extracting the accurate mass at m/z 517.1348, seven peaks were observed and gave MSn spectra. Only one peak was triggered for YG1 and three peaks were triggered for the reference drug YR with normal DDA scan (Table 1). As predicted, malonyl conjugates of O- (58, 33.27 min; 62, 34.97 min) and C-glycosides (50, 29.78 min) of formononetin were the main target compounds. However, some other components showed similar spectra to those of puerarin and different from those of compound 50 (m/z 335.0921, 311.0919, and 293.0814), indicating that the compound had a basic structure of puerarin but did not contain a malonyl group. According to the neutral loss, the acyl was tentatively determined as 3-carboxypropanoyl. Therefore, compound 27 (22.06 min) was characterized as O-(3-caboxypropanoyl)-puerarin, and compounds 45 (27.21 min) and 51 (30.15 min) were tentatively characterized as O-(3-caboxypropanoyl)-daidzin. The peak at 35.69 min (64) also gave predominant fragment ion at m/z 269.0815 in MS/MS spectrum, similar to compound 58 and 62. But the obtained ion produced a totally different decomposition pathway (Figure 5) from that of formononetin. A series of CO (28 Da) were lost instead of CH3 (15 Da) and CH3OH (32 Da), suggesting that the methyl group was attached at a carbon atom directly. The minor ions at m/z 151.0392 and 163.0390 indicated that the methyl was attached at the ring A of isoflavone skeleton. Therefore, compound 64 was tentatively identified as 6″-O-malonyl-C-methyl-daidzin.
Figure 5

MS3 of compound 64

Elaboration of the Compounds Giving Precursor Ions at m/z 519.1138

Seven compounds were extracted with m/z 519.1138. All of them were glycosides of trihydroxyisoflavone according to their MSn spectra. Four peaks at 14.57 (11), 19.58 (22), 26.76 (44), and 29.59 (49) min gave predominant ions in their MS/MS spectra at m/z 271.0607, which then showed different MS3 spectra. The former two showed almost identical fragmentation patterns to those of 7,3′,4′-trihdyroxylisoflavone, and the latter two were obviously similar to 5,7,4′-trihdyroxylisoflavone. Hence, compounds 11 and 22 were tentatively identified as O-malonyl-3′-hydroxydaidzin. At the same time, compounds 44 and 49 were tentatively characterized as O-malonyl-genistin. Three other peaks at 6.23 (2), 12.22 (6), and 24.55 (37) min produced characteristic fragment ion of trihydroxyisoflavone C-glycoside at m/z 313.0714 in their MS/MS spectra. Compounds 2 and 6 showed similar fragmentation patters to those of 4 and 17, respectively, except that all ions were 16 Da larger than those of 4 and 17. Hence, compounds 2 and 6 were tentatively characterized as 4″(6″)-O-malonyl-3′-hydroxy-puerarin. However, compound 37 gave prominent ion at m/z 313.0714, which was different from other C-glycosides. The obtained ion then produced diversity product ions, including the ion at m/z 195.0290, which was considered to be resulted from reaction of RDA. Hence, although there was lack of further information, the compound was assumed to be O-malonyl-5-hydroxy-puerarin, a derivative of genistein.

Elaboration of the Compounds Giving Precursor Ions at m/z 531.1122

Four peaks were found at19.40 (20), 23.71 (34), 32.12 (55), and 34.16 min (61) in the XIC of m/z 531.1122. All of the compounds could not be triggered with the normal DDA scan (Table 1). The [M + H]+ ions at m/z 531.1122 suggested that the chemical formula was C25H23O13, which had one more carbonic atom compared with those producing [M + H]+ ions at m/z 519.1138. However, only compound 61 showed characteristic fragmentation pathways of malonyl glycoside. First, in negative ion mode, the full scans gave obvious [M − H] ion at m/z 529.0981 with the exception of compound 61. Secondly, only the [M + H]+ ion of 61 lost malonyl along with glycosyl (86 + 162 Da) and produced dominant product ion at m/z 283.0599 (C16H11O5). Compared with calycosin, it was assumed to possess a fragment structure of -O-CH2-O-. The hypothesis was supported by the subsequent loss of CH2O (30 Da) in the MS3 spectrum. Therefore, compound 61 was presumed to be pseudobaptigenin O-malonyl-O-glycoside [24]. Compound 34 was characterized as 7,3′,4′-trihydroxyisoflavone O-fumaryl-O-glycoside, whereas compound 55 was assumed to be 5,7,4′-trihydroxyisoflavone O-fumaryl-O-glycoside. Compound 20 was assumed to be O-fumaryl-3′-hydroxypuerarin.

Elaboration of the Compounds Giving Precursor Ions at m/z 533.1296

Twenty compounds were found to produce MSn spectra in the XIC. Unexpectedly, only three compounds were found from YR by the normal DDA scan (Table 1). Obviously, the designed strategy dramatically promoted the recognition ability of the DDA scan. Generally, the malonyl derivatives in this group should present similar characteristic fragment information to that of 3′-methoxylpuerarin, calycosin-7-O-glycoside, or biochanin A, O-/C-glycoside. However, according to the MSn spectra, half of them showed identical characteristic fragment information to that of daidzin or puerarin and the chemical constitution indicating another side chain named maloyl group, which contained one more hydroxymethine compared with malonyl. Accordingly, compounds 7 (12.56 min) and 10 (14.41 min) were characterized as O-moloyl-puerarin and compounds 16 (17.59 min), 18 (17.98 min), 19 (18.37 min), 24 (20.83 min), 25 (21.22 min), 31 (22.91 min), and 32 (23.26 min) were identified as daidzein O-maloyl-O-glycosides.

The compounds at 13 (15.12 min) and 19.49 min (21) produced diagnostic ions of 3′-methoxypuerarin at m/z 309.0766, 327.0872, and 351.0872 in the MS/MS spectrum. Therefore, compounds 13 and 21 was tentatively identified as O-malonyl-3′-methoxypuerarin.

The remaining nine compounds all gave predominant ions at m/z 285.0760. Four of them (28, 22.10 min; 39, 25.94 min; 47, 27.78 min; 48, 28.03 min) then gave almost identical MS3 spectra to MS2 spectrum of calycosin. Therefore, the four compounds were identified as 3′-methoxydaidzein/calycosin O-malonyl-O-glycosides. The other five compounds (46, 27.36 min; 53, 31.47 min; 59, 33.52 min; 60, 33.78 min; 66, 37.55 min) showed similar fragmentation patterns to those of biochanin A, indicating that these compounds were biochanin A O-malonyl-O-glycosides.

Elaboration of the Compounds Giving Precursor Ions at m/z 547.1448

There are 11 compounds that were found in the XIC of m/z 547.1448. Their MS/MS spectra produced predominant ions at m/z 255.0651, 269.0807, or 299.0912, respectively. Five of them (29, 22.33 min; 35, 24.11 min; 38, 25.29 min; 41, 26.25 min; 43, 26.61 min) gave ions at m/z 255.0651 and were tentatively identified as daidzein (O-methylmaloyl)-O-glycosides. The substituent positions could not be determined. Two compounds (56, 32.39 min; 57, 32.59 min) gave base peaks at m/z 269.0807 and were deduced to be O-maloyl-ononin.

Only the compounds (52, 31.40 min; 54, 32.04 min; 63, 35.28 min; 65, 36.29 min) giving base peak ions at m/z 299.0912 (547–248) were plausible malonyl conjugates of isoflavone glycoside. The aglycon could only be tentatively identified as hydroxy-dimethoxy-isoflavone. The substituent positions could not be determined according to the present information. Therefore, compounds 52, 54, 63, and 65 were assumed to be hydroxyl-dimethoxy-isoflavone O-malonyl-O-glycosides.

Elaboration of the Compounds Giving Precursor Ions at m/z 665.1697

Using the normal DDA scan, only two compounds were found from YR and no compound was found from YG1 to trigger the precursor ions at m/z 665.1697 (Table 1). However, 11 components were detected using the designed method. Generally, the compound in this group possessed one more glycosyl compared with O-malonyldaidzin (665 – 503 = 162 Da). According to the MSn spectra, four compounds (1, 5.68 min; 9, 13.96 min; 14, 15.86 min; 15, 16.20 min) were found to be O-malonyl-O-glycosyl puerarin and five compounds (5, 11.51 min; 12, 14.74 min; 30, 22.69 min; 33, 23.49 min; 40, 25.96 min) were found to be O-malonyl-O-glycosyl daidzin, respectively. There were two other compounds also giving [M + H]+ ions at m/z 665.1697. But their fragmentation information was somehow different from that of the nine compounds discussed above. Both of them lost 132 Da (C5H8O4, 665.1697 → 533.1293) first, indicating the present of a xylosyl or apiosyl. The compound at 10.92 min (3) produced obvious fragment ions at m/z 381.0971 and 351.0867 in the MS3 spectrum, which were similar to those of 3′-methoxy mirificin and 3′-methoxy puerarin. Hence, compound 3 was tentatively identified as O-malonyl 3′-methoxy mirificin. However, compound 8 (12.86 min) was tentatively characterized as 2″-O-maloyl-mirificin.

Conclusion

Malonyl glycoside conjugates of isoflavone are ubiquitous in leguminous TCMs, including those originating from Pueraria, and play an important role in metabolism and biosynthesis of the secondary metabolites. The conjugates in Gegen usually present in low, sometimes even trace, amounts and are always covered by other abundant components, which make them hard to detect in the normal analysis method. Interestingly, the malonyl glycosides were found to be particularly labile in ESI negative mode using LTQ Orbitrap Velos Pro mass spectrometer. A specific method was developed in this study for screening and identifying the malonyl conjugates of isoflavone glycoside using ion mapping method combined with precursor mass list and dynamic exclusion. The established method was applied to analyze two Gegen samples. A total of 66 compounds were characterized from the samples, and 43 of them were malonyl conjugates of isoflavone glycoside (Table 1, Supplementary Figure 5S, Supplementary Table 1S). However, only 20 compounds could be characterized with the normal method. The designed method was proven to be specific and efficient for screening and identifying the malonyl conjugates in Gegen. Also, the method could be used for analysis of malonyl glycosides in other TCMs or metabolites in biosamples.

In TCM analysis, hundreds and even thousands of compounds could be detected with the enhanced ability of the LC-MS technology. To comprehensively characterize the compounds is usually impossible using a normal DDA method. Sometimes, aiming for the special types of components is essential and more efficient than global analyzing the miscellaneous constituents. The designed specific method is also useful for in vivo investigation of metabolism in complex matrices.

Notes

Acknowledgments

The authors acknowledge financial support for this work by the National Natural Science Foundation of China (81373965, 81001630), the Major Project of Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-YW-R-166), the 12th Five-Year National Science and Technology Support Program (2012BAI29B06), and the National Science and Technology Major Project “Key New Drug Creation and Manufacturing program” (number: 2011ZX09307-002-03) of the People’s Republic of China.

Supplementary material

13361_2015_1240_MOESM1_ESM.docx (694 kb)
ESM 1 (DOCX 694 kb)

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Copyright information

© American Society for Mass Spectrometry 2015

Authors and Affiliations

  • Min Yang
    • 1
  • Zhe Zhou
    • 2
  • Shuai Yao
    • 1
  • Shangrong Li
    • 1
  • Wenzhi Yang
    • 1
  • Baohong Jiang
    • 1
  • Xuan Liu
    • 1
  • Wanying Wu
    • 1
  • Hua Qv
    • 1
  • De-an Guo
    • 1
    Email author
  1. 1.National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia MedicaChinese Academy of SciencesShanghaiChina
  2. 2.ThermoFisher Scientific (China) Co., Ltd.ShanghaiChina

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