Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry

Analytica Chimica Acta 598 (2007) 110–118

Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry Peiying Shi, Qing He, Yue Song, Haibin Qu ∗ , Yiyu Cheng ∗ Pharmaceutical Informatics Institute, Zhejiang University, Hangzhou 310027, PR China Received 3 May 2007; received in revised form 11 July 2007; accepted 11 July 2007 Available online 14 July 2007

Abstract Flavonoid O-diglycosides are important bioactive compounds from genus Citrus. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization (ESI) with loop injection. For the flavonoid O-rutinosides, [M − H − 308]− ion was typically observed in the MS2 spectrum, suggesting the loss of a rutinose. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M − H − 120]− ion in the MS2 spectrum, which was a common feature of all the flavonoid O-neohesperidosides. The previous literature for naringin located the loss of 120 Da to the glycan part, whereas the present study for naringin had shown that the [M − H − 120]− ion was produced by a retro-Diels-Alder reaction in ring C, and this fragmentation pattern was confirmed by the accurate mass measurement using an orthogonal time-of-flight mass spectrometer. Combined with high performance liquid chromatography (HPLC) and diode array detection (DAD), the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of two Chinese medicines derived from genus Citrus, namely Fructus aurantii and F. aurantii immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by multistage mass spectrometry (MSn ) spectra, 13 flavonoid O-glycosides in F. aurantii and 12 flavonoid O-glycosides in F. a. immaturus were identified rapidly. © 2007 Elsevier B.V. All rights reserved. Keywords: Flavonoid O-diglycosides; Ion trap; Time-of-flight; Mass spectrometry

1. Introduction Flavonoid constituents are abundant in medicinal plants from genus Citrus. Until now, more than 60 flavonoids have been isolated from genus Citrus and structurally elucidated [1]. Among these compounds, the O-diglycosides are a dominant category, and their structures are usually characterized by the linkage of either ␤-neohesperidose (2-O-␣-l-rhamnopyranosyl␤-d-glucopyranose) or ␤-rutinose (6-O-␣-l-rhamnopyranosyl␤-d-glucopyranose) to the flavonoid skeleton through the C-7 hydroxyl group [2]. The strong antioxidant activities of flavonoid

∗

Corresponding author. Tel.: +86 571 87951138; fax: +86 571 87951138. E-mail addresses: [email protected] (H. Qu), [email protected] (Y. Cheng). 0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.07.027

O-diglycosides have been indicated by many pharmacological studies [3–6]. However, the flavonoid O-diglycosides derived from genus Citrus often exist as isomers, resulting in confusion in the structural identification. Consequently, rapid and accurate distinguishing of these isomers is a challenge to researchers. Recently, ion trap mass spectrometry has become a powerful approach to the rapid and veritable investigation of these isomers because of its tandem MS/MS capabilities. Brodbelt and her group have paid considerable attention to developing mass spectrometric methods to differentiate isomeric flavonoid Odiglycosides based on metal complexation [7–11]. Claeys and co-workers have initiated a series of studies on MS analysis in the positive and negative ion modes of the isomeric flavonoid O-diglycosides [12,13]. Although characterization of flavonoid O-diglycosides in negative electrospray ionization by ion trap mass spectrometry has been discussed, the elucidation of some

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product ions in MS/MS spectrum is only tentatively deduced and need further confirmation. Up to now, there have been many reports dealing with the fragmentation characteristics of isomers in medicinal plant extracts, such as flavonoids [14], saponins [15] and alkaloids [16] using combination of ion trap mass spectrometry and timeof-flight mass spectrometry. Rich structural information can be obtained through collision-induced dissociation (CID) in the course of MSn analysis. Elemental composition of both precursor and product ions can be acquired from the high-resolution mass spectrometry (HRMS) analysis. Therefore, the structures of product ions obtained from the MS/MS experiments can be confirmed. Fructus aurantii and F. aurantii immaturus are two widely used Chinese medicines derived from genus Citrus, and have been well documented in the China Pharmacopeia as drugs to activate vital energy and circulation, eliminate phlegm and remove food retention [17]. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization. Accurate mass measurements were achieved with an orthogonal time-of-flight mass spectrometer to support the hypothesis of fragmentation pathways. Combined with high performance liquid chromatography and diode array detection, the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of F. aurantii and F. a. immaturus. 2. Experimental 2.1. Reagents and materials HPLC-grade acetonitrile, methanol (Merck KGaA, Darmstadt, Germany) and formic acid (TEDIA, Fairfield, OH, USA) were used for HPLC analysis. Deionized water was purified by a Milli-Q system (Millipore, Bedford, MA, USA). Reference compounds including hesperidin (R1), neohesperidin (R2), narirutin (R3), naringin (R4), diosmin (R5) and poncirin (R6) were isolated from F. aurantii, and their structures were elucidated by NMR spectroscopic analysis. Purities of all the reference compounds were greater than 98% according to HPLC analysis based on a peak area normalization method. F. aurantii and F. a. immaturus were purchased from Hangzhou traditional Chinese medicine factory (Zhejiang, China) and authorized by Dr. Qing He, one of the authors. 2.2. Standard solutions and preparation of sample solutions Methanolic stock solutions containing reference compounds with known concentrations (50–150 ␮g mL−1 ) were prepared and stored in a refrigerator (4 ◦ C) until use. The samples were dried and ground into powder (40 mesh). An aliquot of 1 g of each sample was accurately weighted and ultrasonically extracted with 50 mL of methanol for 30 min. The extracts were centrifuged at 10 000 rpm for 10 min and aliquots

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of 10 ␮L of each sample were injected for LC/MS or LC/MSn analysis. 2.3. Mass spectrometric analysis of reference compounds 2.3.1. MSn analysis of reference compounds A Finnigan LCQ Deca XPplus ion trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with an ESI interface and an ion trap mass analyzer was used to carry out the MS and MSn analysis. A syringe pump was used for the direct loop injections of reference compound solutions, and the flow rate was set at 5 ␮L min−1 . The operating parameters in the negative ion mode were as follows: collision gas, ultrahighpurity helium (He); nebulizing gas, high purity nitrogen (N2 ); ion spray voltage, −4.5 kV; sheath gas (N2 ), five arbitrary units; capillary temperature, 275 ◦ C; capillary voltage, −15 V; tube lens offset voltage, −30 V. The collision energy for CID was between 25% and 35% of maximum, and the isolation width of precursor ions was 2.0 Th. 2.3.2. HRMS analysis of neohesperidin and naringin Two reference compounds, neohesperidin and naringin, were analyzed using an orthogonal time-of-flight mass spectrometer (Agilent Crop., Santa Clara, CA, USA) in negative ion mode. The parameters were as follows: mass range, m/z 100–3000; the flow rate of drying gas (N2 ), 9.0 L min−1 ; drying gas temperature, 310 ◦ C; nebulizer, 35 psig; capillary voltage, 4000 V; fragmentor, 350 V; skimmer voltage, 60 V; octopole DC1, 37 V; octopole rf, 250 V. 2.3.3. Ion nomenclature The nomenclature proposed by Domon and Costello [18] for glycoconjugates was adopted to denote the product ions. Ions containing the aglycone are labeled k,l Xj , Yj and Zj , where j is the number of the interglycosidic bond broken, counted from the aglycone, and the superscripts k and l indicate the cleavages within the carbohydrate rings. The glycosidic bond linking the glycan part to the aglycone is numbered 0. 2.4. LC/MS 2.4.1. HPLC conditions LC analysis was performed on an Agilent 1100 HPLC system (Agilent, Waldbronn, Germany) including a binary pump, a diode array detector, an auto-sampler and a column compartment. The separation was carried out on a Zorbax SBC18 column (5 ␮m, 4.6 mm × 250 mm, Agilent). The mobile phase consisted of acetonitrile (A) and water–formic acid (100:0.1, v/v) (B). A gradient program was carried out as follows: 0–50 min, start with 10% B, then linearly increase to 30%; 50–65 min, linearly increase to 55%; 65–75 min, linearly increase to 60%; 75–90 min, linearly increase to 95%. The flow rate was 1 mL min−1 , and the temperature of column oven was 30 ◦ C. The UV spectra were recorded from 190 nm to 400 nm.

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2.4.2. ESI-MS analysis The LC flow was introduced with a splitter to provide a 200 ␮L min−1 flow to the MS system. All the parameters were the same as those described in MSn experiments except that the capillary temperature was set at 350 ◦ C, sheath gas (N2 ) flow rate at 30 arbitrary units and auxiliary gas (N2 ) flow rate at 10 arbitrary units. Full scan data acquisition was performed from m/z 100 to m/z 1500 in MS scan mode. A data-dependent program was used so that the three most abundant ions in each MS scan were selected and subjected to tandem mass spectrometry (MSn , n = 2–3) analysis. The collision energy for CID was adjusted to 30% of maximum. 3. Results and discussion 3.1. MSn and HRMS analysis of reference compounds The MS data of the references and their major fragments in MSn spectra were summarized in Table 1, and the structures of the references were elucidated in Fig. 1. All the reference compounds exhibited the [M − H]− ions, and the precursor ions dissociated at low CID energy and gave the fragments. 3.1.1. Characterization of flavonoid O-rutinosides R1 and R3 produced [M − H − 308]− ion in the MS2 spectrum, suggesting the loss of a rutinose (Fig. 2a and c). Another flavonoid O-rutinoside, diosmin (R5), also showed similar fragTable 1 The MSn (n = 2–3) data of the reference compounds Compounds

[M − H]−

ESI-MSn m/z (% base peak)

Hesperidin (R1)

609

MS2 [609]: 609(16), 301(100) MS3 [609 → 301]: 301(28), 286(100), 283(48), 258(19), 257(45), 242(75), 199(8), 125(8)

Neohesperidin (R2)

609

MS2 [609]: 609(33), 489(18), 403(6), 343(20), 325(6), 301(100) MS3 [609 → 489]: 489(46), 301(100) MS3 [609 → 301]: 301(13), 286(100), 283(49), 258(21), 257(41), 242(68), 199(7), 125(6)

Narirutin (R3)

579

MS2 [579]:

579(28), 271(100) MS3 [579 → 271]: 177(17), 151(100)

Naringin (R4)

579

MS2 [579]: 579(36), 459(100), 373(7), 313(13), 271(26), 235(6) MS3 [579 → 459]: 459(21), 441(26), 357(64), 339(18), 271(35), 235(100), 211(6), 205(9), 193(6), 151(7)

Diosmin (R5)

607

MS2 [607]: 607(14), 299(100), 284(9) MS3 [607 → 299]: 299(24), 284(100)

Poncirin (R6)

593

MS2 [593]: 593(32), 473(23), 431(7), 387(11), 327(30), 285(100) MS3 [593 → 285]: 285(23), 270(81), 243(100), 241(16), 226(7), 217(6), 164(60) MS3 [593 → 473]: 473(50), 285(100)

Fig. 1. Structures of identified compounds from the extracts of Fructus aurantii and F. aurantii immaturus.

mentation pattern, but only a low intensity ion at m/z 284 was observed in its MS2 spectrum (Fig. 2e), which was defined as [M-H-308-CH3 ·]− ion due to the dissociation of [M − H − 308]− ion. 3.1.2. Characterization of flavonoid O-neohesperidosides The spectrum of R2 was shown in Fig. 2b. Y0 − ion corresponded to the base peak. The [M − H − 120]− ion was attributed to 0,2 X0 − ion, which accorded with the reported data [13] and was confirmed by the MS3 profile, where the ion at m/z 301 was produced. Further loss of the terminal rhamnose residue yielded a 0,2 X0 Y1 − ion at m/z 343 and a 0,2 X0 Y1−w − ion at m/z 325, which were also in good agreement with the literature [19].

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Fig. 2. MS2 spectra of the [M − H]− ions of hesperidin (a), neohesperidin (b), narirutin (c), naringin (d), diosmin (e) and poncirin (f).

An ion was also observed at m/z 403, which could be defined as or 2,4 X0 Y1 − ion (Scheme 1). Compared with R2, the same fragments of [M − H − 120]− ion, 0,2 X0 Y1 − ion and 1,3 X0 − or 2,4 X0 Y1 − ion were also observed in the MS2 spectrum of R4 (Fig. 2d), which was consistent with the literature [13]. However, the intensity of [M − H − 120]− ion was extremely different between the MS2 spectra of R2 and R4, as well as the MS3 data (Fig. 3). Although the previous literature for naringin located the loss of 120 Da to the glycan part [12,13,19], it was uncertain that the [M − H − 120]− ion was the same as 0,2 X0 − ion, because of the two differences as mentioned above. Under low-energy CID conditions, a retro-Diels-Alder fragmentation in ring C involving a cleavage of bonds 1 and 3 of the aglycone of R2 was very easy to occur [20], so the fragmentation of loss 120 Da of R4 might be produced in ring C rather than in the glycan part. This 1,3 X − 0

could be attributed to the existence of a hydroxyl group linked to 4 position of ring B. In order to confirm this hypothesis, the accurate mass measurement for both precursor ions and product ions, [M − H − 120]− ions of R2 and R4, was carried out by the HRMS analysis with the accuracy less than 10 ppm (Table 2). The results of R2 accorded with the literature [19] and the tentative proved that the retro-Diels-Alder fragmentation of loss 120 Da of the [M − H]− ion of R4 was the main fragmentation pattern of these flavonoid O-neohesperidosides (Scheme 2). Poncirin (R6) was also a flavonoid O-neohesperidoside as R2 and R4, and the fragmentation profiles exhibited similarities in Y0 − ion, 0,2 X0 − ion, 0,2 X0 Y1 − ion and 1,3 X0 − or 2,4 X0 Y1 − ion (Fig. 2f). However, a low intensity ion at m/z 431 due to the loss of 162 Da from precursor ion was observed in the MS2 spectrum of R6. It was assigned to the glucose and has not been reported in the previous literature.

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Scheme 1. Proposed MS2 pathway for neohesperidin.

Fig. 3. MS3 spectra of the [M − H − 120]− ions of neohesperidin (a) and naringin (b).

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Table 2 HRMS data from precursor and product ions of neohesperidin and naringin Neohesperidin

Naringin

Elemental composition Theoretical value Measured value Mass accuracy (ppm)

C28 H33 O15 609.1824 609.1832 1.1581

C27 H31 O14 579.1719 579.1719 −0.0514

[M − H − 120]− Elemental composition Theoretical value Measured value Mass accuracy (ppm)

C24 H25 O11 489.1402 489.1393 −1.9127

C19 H23 O13 459.1144 459.1144 −0.0322

[M − H]−

3.2. HPLC-DAD/ESI-MSn analysis of the extracts of F. aurantii and F. a. immaturus The HPLC-DAD chromatogram and total ion current (TIC) chromatogram in negative ion mode of the extract of F. aurantii were shown in Fig. 4, and most constituents were well separated and responded in full scan mode. The data of retention time (tR ), molecular weight and the maximal UV wavelength of the constituents detected in the extract of F. aurantii were listed in Table 3. Due to the typical absorption pattern of ring B, cinnamoyl system and ring A, benzoyl system, the flavonoids and their various modified forms were rapidly confirmed by their UV characteristic profiles. Flavonoid O-diglycosides displayed a strong absorption at 284 nm and a weak absorption at 320–330 nm simultaneously [1]. According to the typical UV absorption and fragmentation pattern proposed above, compounds 5, 7–10, 13, 16 and 17 were identified as O-diglycosyl flavonones, compounds 11 and 12 as O-diglycosyl flavones, compounds 2, 3 and 4 as O-triglycosyl flavanones. Their MSn data were listed in Table 4 and most of their structures were shown in Fig. 1. 3.2.1. Characterization of O-diglycosyl flavonones Compounds 8, 9, 10, 13 and 17 were confirmed to be narirutin, naringin, hesperidin, neohesperidin and poncirin respectively, by comparing their MS, MSn data, retention time and UV spectra with those of the five reference compounds.

Fig. 4. HPLC-DAD chromatogram of extracts of F. aurantii (a); TIC chromatogram of extracts of F. aurantii from HPLC-(-) ESI-MS (b).

Compound 16 gave a [M − H]− ion at m/z 593. Only the product ion [M − H − 308]− was observed in its MS2 spectrum, suggesting the loss of a rutinose. The MS3 characteristic profile of [M − H − 308]− ion was identical with that of specified aglycone ion of R6, which demonstrated that compound 16 and R6 had the same aglycone. From the above results, compound 16 was identified as didymin [21]. Furthermore, the retention time of hesperidin was less than that of didymin, which conformed to the general rule that an increase in the number of free hydroxyl groups resulted in shorter HPLC retention time [2]. Thus, the elution order of hesperidin and didymin further supported our conclusion. The [M − H]− ion of compounds 5 and 7 was m/z 595, an increase of 16 Da compared to those of narirutin and naringin,

Scheme 2. Proposed major MS2 pathway for naringin and neoeriocitrin.

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Table 3 Peak assignments for analysis of extracts from F. aurantii and F. a. immaturus Peak no.

a b

UV λmax (nm)

tR (min)

Identification

1

14.9

Unknowna,b

593 1187 [2M − H]−

270, 336

2

16.72

O-Triglycosyl naringenina,b

741 [M − H]− 787 [M − H + HCOOH]−

–

3

18.55

O-Triglycosyl naringenina,b

741 [M − H]− 787 [M − H + HCOOH]−

–

4

22.87

O-Triglycosyl naringenina,b

741 [M − H]− 787 [M − H + HCOOH]−

284, 326(low)

5

23.47

Eriocitrina,b

595 [M − H]− 641 [M − H + HCOOH]− 1191 [2M − H]−

284, 332(low)

6

24.09

Unknowna,b

649 [M − H]− 695 [M − H + HCOOH]− 1299 [2M − H]−

–

7

25.51

Neoeriocitrina,b

595 [M − H]− 641 [M − H + HCOOH]− 1191 [2M − H]−

284, 330(low)

8

29.28

Narirutina,b

579 [M − H]− 625 [M − H + HCOOH]− 1159 [2M − H]−

284, 330(low)

9

31.44

Naringina,b

579 [M − H]− 625 [M − H + HCOOH]− 1159 [2M − H]− 1205 [2M − H + HCOOH]−

284, 328(low)

10

32.98

Hesperidina,b

609 [M − H]− 655 [M − H + HCOOH]− 1219 [2M − H]−

284, 324(low)

11

33.46

Diosmina

607 [M − H]− 653 [M − H + HCOOH]− 1261 [2M − H + HCOOH]−

–

12

34.55

Chrysoeriol 7-␤-rutinosidea,b

607 [M − H]− 653 [M − H + HCOOH]− 1215 [2M − H]− 1261 [2M − H + HCOOH]−

–

13

35.1

Neohesperidina,b

609 [M − H]− 655 [M − H + HCOOH]− 1219 [2M − H]− 1265 [2M − H + HCOOH]−

284, 324(low)

14

37.45

Unknowna,b

693 [M − H]− 1387 [2M − H]−

–

15

39.65

Unknowna,b

723 [M − H]−

–

16

46.1

Didymina,b

[M − H]−

593 639 [M − H + HCOOH]−

–

17

48.15

Poncirina,b

593 [M − H]− 639 [M − H + HCOOH]− 1233 [2M − H + HCOOH]−

284, 326(low)

18

49.17

Unknowna

709 [M − H]− 1419 [2M − H]−

–

19

55.5

Unknowna,b

723 [M − H]− 1447 [2M − H]−

288

Compounds detected from F. aurantii. Compounds detected from F. a. immaturus.

(−) ESI-MS m/z [identity] [M − H]−

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Table 4 UV absorption peaks and MSn data in negative ion mode of compounds observed from the extracts of F. aurantii Peak no.

HPLC/ESI-MSn m/z (% base peak)

1

MS2 [593]: 575(7), 503(28), 473(100), 383(18), 353(30) MS3 [593 → 473]: 473(29), 383(13), 353(100)

2

MS2 [741]: 579(13), 433(100) MS3 [741 → 579]: 271(100) MS3 [741 → 433]: 271(100)

3

MS2 [741]: 741(6), 621(12), 579(100) MS3 [741 → 579]: 579(24), 459(100), 313(15), 271(28), 235(6)

4

MS2 [741]: 742(62), 621(100), 459(11), 271(11) MS3 [741 → 621]: 621(100), 603(9) MS3 [741 → 271]: 271(94), 177(16), 165(20), 151(100) MS3 [741 → 459]: 459(6), 357(6), 339(26), 315(6), 271(100), 235(7)

5

MS2 [595]: 287(100) MS3 [595 → 287]: 287(3), 269(3), 169(1), 151(100), 135(2), 125(2), 107(1)

6

MS2 [649]: 631(9), 605(100), 461(18), 443(55), 385(8), 209(8) MS3 [649 → 605]: 605(100), 587(13), 562(7), 461(14), 443(77), 399(11), 385(26)

7

MS2 [595]: 595(15), 459(100), 287(7), 235(8) MS3 [595 → 287]: 287(1), 286(1), 269(4), 151(100), 135(2), 125(1) MS3 [595 → 459]: 459(9), 441(26), 357(64), 339(11), 271(22), 235(100), 217(8), 211(10), 205(12), 193(11), 151(15)

8

MS2 [579]: 271(100) MS3 [579 → 271]: 271(82), 177(23), 151(100)

9

MS2 [579]: 579(43), 459(100), 313(14), 271(90), 235(7) MS3 [579 → 271]: 271(86), 177(15), 165(9), 151(100) MS3 [579 → 459]: 459(6), 441(27), 357(78), 339(27), 271(44), 235(100), 211(11), 205(16), 193(8), 151(13)

10

MS2 [609]: 301(100) MS3 [609 → 301]: 301(100), 286(15), 283(9), 257(8), 242(9)

11

MS2 [607]: 607(70), 299(100), 284(10) MS3 [607 → 299]: 299(100), 284(9)

12

MS2 [607]: 607(7), 299(100), 284(10) MS3 [607 → 299]: 299(100), 284(39)

13

MS2 [609]: 609(59), 489(21), 447(6), 403(6), 343(22), 325(7), 301(100) MS3 [609 → 301]: 301(100), 286(16), 283(9), 257(7), 242(9) MS3 [609 → 489]: 489(44), 301(100)

14

MS2 [693]: 633(15), 589(13), 565(100), 489(7), 471(10), 445(6), 401(6), 395(10) MS3 [693 → 565]: 403(100), 345(16), 327(17), 165(11)

15

MS2 [723]: 661(9), 621(30), 579(100) MS3 [723 → 579]: 579(14), 459(100), 373(7), 313(15), 271(31), 235(9)

16

MS2 [593]: 285(100) MS3 [593 → 285]: 285(100), 270(9), 243(11), 164(7)

17

MS2 [593]: 593(99), 473(27), 431(12), 387(14), 369(5), 327(34), 309(2), 285(100) MS3 [593 → 285]: 285(100), 270(13), 243(11), 164(8) MS3 [593 → 473]: 473(100), 285(95)

18

MS2 [709]: 647(10), 607(40), 565(100), 403(16) MS3 [709 → 565]: 403(100)

19

MS2 [723]: 607(9), 417(100), 403(6) MS3 [723 → 417]: 417(47), 402(100), 359(10)

which indicated that compounds 5 and 7 might be the hydroxyl derivatives of narirutin and naringin. The ion at m/z 287 was the only peak observed in MS2 spectrum of compound 5 after the neutral loss of a rutinose. In the MS2 spectrum of compound 7, the product ion at m/z 459 after cleavage of ring C was observed like that of naringin, and the MS3 profile of the ion at m/z 459

from compound 7 and naringin was identical, which could be inferred that the hydroxyl group linked to ring B. The same as characteristic pattern of naringin, compound 7 exhibited similar fragmentation profile (Scheme 2). The aglycone of compounds 5 and 7 gave identical characteristic ions, and the ions were consistent with those in the MS2 spectrum of eriodictyol [20].

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Therefore, compounds 5 and 7 were confirmed to be eriocitrin and neoeriocitrin, respectively. 3.2.2. Characterization of O-diglycosyl flavones Compound 11 was identified as diosmin by comparing the retention time and MSn data with those of the reference compound. However, the fragments of compound 12 were the same as those of diosmin after a series of CID fragmentations. In comparison with the present flavonoids isolated from genus Citrus [1], compound 12 was tentatively identified as chrysoeriol 7␤-rutinoside, where the methyl group linked to 4 position of ring B. The elution order of the two compounds was consistent with the literature that the compound containing 3 -hydroxy-4 methoxy substitution pattern eluted ahead of the one including 4 -hydroxyl-3 -methoxyl substitution pattern [2], which also supported our hypothesis. 3.2.3. Characterization of O-triglycosyl flavanones In the MS2 spectrum of [M − H]− ion of compound 2, two major ion peaks, [M − H − 162]− and [M − H − 308]− ions were detected and produced product ion at m/z 271 in MS3 spectrum, suggesting the presence of a hexose and a rutinose substituents. In the MS2 spectrum of [M − H]− ion of compound 3, [M − H − 162]− ion corresponded to the base peak. After further dissociation, the fragmentation profile was the same as that of naringin, indicating the presence of a hexose and a neohesperidose substituents. Therefore, compounds 2 and 3 were tentatively identified as O-triglycosyl naringenin. Similarly, compound 4 was tentatively identified as another Otriglycosyl naringenin. 4. Conclusion In this paper, the MSn characteristics of flavonoid O-diglycosides from genus Citrus were investigated and fragmentation patterns of flavonoid O-diglycosides in negative electrospray ionization were proposed in detail. The fragmentation pattern of retro-Diels-Alder reactions in ring C of naringin was proposed and confirmed by HRMS analysis. An HPLC-DAD/ESI-MSn method was developed for the analysis of flavonoid O-glycoside constituents in two Chinese medicines, F. aurantii and F. a. immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular

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