quadrupole time-of-flight mass spectrometry

Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

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Effective 2D-RPLC/RPLC enrichment and separation of micro-components from Hedyotis diffusa Willd. and characterization by using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry Cunman Li a,1 , Yanyan Zhao b,1 , Zhimou Guo c , Xiuli Zhang c , Xingya Xue c , Xinmiao Liang c,∗ a

Hebei Research Centre of Analysis and Testing, Hebei University of Science and Technology, Shijiazhuang 050018, China Pharmacy College, Dalian Medical University, Dalian 116044, China c Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China b

a r t i c l e

i n f o

Article history: Received 14 October 2013 Received in revised form 12 March 2014 Accepted 13 June 2014 Available online 9 July 2014 Keywords: Micro-component UPLC–DAD/Q-TOF MS 2D-RPLC/RPLC Click OEG stationary phase Hedyotis diffusa Willd.

a b s t r a c t An effective method aiming at enrichment and analysis of micro-components in traditional Chinese medicine (TCM) was developed. One fraction (fraction E) from the extract of Hedyotis diffusa Willd. was selected as test sample, which was isolated by using the XAD-4 macroporous resin. To study the microcomponents, a two-dimensional reverse-phase liquid chromatography (2D-RPLC/RPLC) method was developed, comprising Click OEG and C18 stationary phases as the first and second dimensions, respectively. Of the eight sub-fractions isolated from the first dimension, three sub-fractions (fractions II–IV) containing micro-components were further separated with the second dimension. The 2D-RPLC/RPLC system was proved to possess high orthogonality. Furthermore, the micro-components were characterized by using ultra-performance liquid chromatography–diode array detector/quadrupole time-of-flight mass spectrometry (UPLC–DAD/Q-TOF MS) with electrospray ionization (ESI) source. With the optimized separation and characterization method, a large number (>400) of micro-components were enriched and detected from the extracts of H. diffusa Willd., the majority of which has not been isolated from the herb before. Among these isolated micro-components, 38 compounds involving 24 phenylpropanoids, 7 flavonoids and 7 iridoid glucosides (IGs), were identified or tentatively identified from the H. diffusa extracts on the basis of spectral data of the authentic standards and the fragmentation characteristics information available in literatures. The proposed method made it possible to effectively screen and analyze the micro-components in TCMs or other complex natural medicines. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Traditional Chinese medicine (TCM) is characterized with high complexity, including large number of compounds that vary in their physical and chemical properties as well as their contents. Microcomponents are one of the most important groups in TCM, some of which show high bioactivities in spite of their very low contents [1]. Therefore, isolation and characterization of micro-components from TCMs is of great importance during the research work for

∗ Corresponding author. Tel.: +86 41184379519; fax: +86 41184379539. E-mail address: [email protected] (X. Liang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jpba.2014.06.020 0731-7085/© 2014 Elsevier B.V. All rights reserved.

novel drugs. However, difficulty often encountered in the process of isolation and characterization of micro-components from extracts of TCMs is due to the following reasons: (1) micro-components are difficult to be detected due to the low correspondence signal when analysis with UV or MS detection. Moreover, micro-components often suffer from ion-suppression effect brought by their highabundance counterparts during MS analysis. (2) Micro-components are often co-eluted with major components during chromatographic isolation. The inefficient isolation brings more difficulties to detect and characterize micro-components. To solve the problems, efforts should be made to improve the chromatographic isolation efficiency and enhance the signal response of micro-components. Compared with the singledimensional liquid chromatography, two-dimensional liquid

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C. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

chromatography (2D-LC) is more suitable to be applied in the isolation of micro-components due to the greatly increased peak capacity. The 2D-LC methods incorporating two dimensions with different separation mechanisms were increasingly applied in separation of compounds from TCM [2–4]. To develop an efficient 2D-LC method, the stationary phases applied in both dimensions should be selected. Click OEG (oligo (ethylene glycol)) stationary phase was synthesized in our group before [5], and it was proved with different separation selectivity from that of traditional C18 under reverse-phase liquid chromatography (RPLC) mode [6]. Wang et al. found that simple phenylpropanoids and lignans could be enriched with Click OEG stationary phase, and more than 20 simple phenylpropanoids could be isolated by using the Click OEG stationary phase and characterized from the extract of Forsythia suspensa [7]. But to the best of our knowledge, micro-components as the main research object have been scarcely studied so far in TCMs or other complex natural medicines. Based on the high orthogonality between Click OEG and C18 stationary phases, coelution of micro-components with their counterparts could be reduced, and thus the micro-components are expected to be better isolated from their counterparts. Hedyotis diffusa Willd. is one of the widely used TCMs, which is proved with many bioactivities, such as antioxidant activity [8], human neutrophil elastase inhibitory effect [9], and anti-cancer activity [10]. Up to now, three major classes of compounds are reported from the extract of H. diffusa, including iridoid glucosides (IGs), flavonoids, and anthraquinones. However, the chemical components especially the micro-components of H. diffusa are far from clarity, which is possibly due to the inefficient isolation and characterization methods. In order to better understand the bioactivity of compounds from the extract of H. diffusa, efforts should be made to improve the separation and identification methods. Liquid chromatography/high-resolution mass spectrometry (LC/HRMS) such as ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) [11], is a very useful approach in separation and identification of compounds from complex samples. To the best of our knowledge, so far only one literature was related to analysis of the components in H. diffusa by using UPLC/Q-TOF MS [12], but not to mention analyzing micro-components. Considering the high resolution and low detection limit of UPLC/Q-TOF MS method, it is suitable for analysis of micro-components from extracts of H. diffusa. In this work, Click OEG stationary phase was used as the first dimension of 2D-RPLC/RPLC method for the enrichment of microcomponents from the extract of H. diffusa. The fractions comprising micro-components collected from Click OEG stationary phase were further isolated on the traditional C18 stationary phase and characterized by using UPLC/Q-TOF MS.

2. Experimental 2.1. Reagents and materials Acetonitrile and methanol (HPLC-grade) were purchased from Fisher Scientific (Loughborough, Great Britain), and formic acid (HPLC-grade) was purchased from Acros Organics (New Jersey, USA). Water was purified with a Milli-Q water-purification system (Millipore, Bedford, MA, USA), which was used for separation and UPLC–DAD/Q-TOF analysis. The aerial parts of H. diffusa were collected from Hengdong County, Hunan province (China). The herb was authenticated by Institute of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences. The voucher specimens were deposited at

Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China.

2.2. Preparation of micro-component extract The procedure for preparation of micro-component extracts was shown in Fig. 1. Dried and powdered H. diffusa (2.0 kg) was refluxed twice with water, each for 1 h. After filtration, the filtrate was dried under vacuum at 60 ◦ C. The resulting aliquot (200 g) was dissolved in 1.2 L water–ethanol (20:80, v/v) solution and placed for 24 h and then filtered, eliminating the water-soluble large molecular impurities. The filtrate was dried by using a rotary evaporator under reduced pressure at 60 ◦ C and 26.7 g residue (fraction A) was obtained. Subsequently, the fraction A was loaded onto an XAD-4 macroporous resin column and eluted successively with H2 O, 10%, 30%, and 95% ethanol–water (v/v) solution. As a result, 6.6 g residue (fraction B) from water solution, 8.8 g residue (fraction C) from 10% ethanol–water solution (v/v), 4.3 g residue (fraction D) in 30% ethanol–water (v/v) solution and 6.0 g residue (fraction E) in 95% ethanol–water (v/v) solution were obtained, respectively. In order to enrich the micro-components and simplify the compositions, fraction E was separated by using a preparative reversed-phase liquid chromatography with UV detector (prep-RPLC, Dalian Elite Analytical Instrument Co., China). Chromatograms were monitored at a wavelength of 254 nm. Separation was performed on Click OEG column (500 mm × 30 mm i.d., 10–20 ␮m). The linear gradient consisted of methanol (A) and water (B) with a flow rate of 200.0 mL/min. The optimized chromatographic gradient was: 5% A at 0–5 min, 5–10% A at 5–10 min, 10–30% A at 10–30 min, 30–100% A at 30–31 min, 100% A at 31–35 min. The chromatographic effluent was collected based on the UV signal, and eight fractions (I–VIII) were collected. Of the eight fractions, fractions II, III, IV were studied as the micro-component samples for UPLC–DAD/Q-TOF MS analysis. An aliquot (10 mg) of each micro-component sample was dissolved in water (10 mL) and filtered through a membrane filter (0.22 ␮m) prior to UPLC/Q-TOF MS analysis.

2.3. UPLC–DAD/Q-TOF MS analysis of micro-components UPLC–DAD/Q-TOF MS analysis was performed in both negative and positive ion modes with a Waters Acquity UPLC coupled with a Q-TOF Premier, a quadrupole and orthogonal acceleration time-of-flight tandem mass spectrometer (Milford, MA, USA). High purity nitrogen was used as the nebulizer and auxiliary gas, and argon was used as the collision gas. The electrospray ionization (ESI) capillary voltage was set at 2.5 kV. The source and desolvation temperatures were set at 120 ◦ C and 350 ◦ C, respectively. The nitrogen desolvation and cone gas flow rates were set at 800 L/h and 50 L/h, respectively. The sample cone voltage was set at 35 V, and the collision energy was set at 10–20 eV. The spectra scan range was from m/z 50 to 1000. For on-line analysis of the extracts of H. diffusa, an Acquity Shield RP C18 UPLC column (100 mm × 2.1 mm i.d., 1.7 ␮m, Waters corporation) was used, with a flow rate of 0.25 mL/min. The column temperature was set at 25 ◦ C. The elution system consisted of acetonitrile (A) and 0.1% formic acid aqueous solution (v/v) (B). The gradient programs were: (1) fraction II: 3% A at 0–3 min, 3%–10% A at 3–13 min, 10–15% A at 13–38 min, 15–25% A at 38–58 min, 25–40% A at 58–63 min, 40–95% A at 63–65 min; (2) fraction III: 3–10% A at 0–13 min, 10–15% A at 13–38 min, 15–25% A at 38–58 min, 25–40% A at 58–63 min, 40–95% A at 63–65 min; (3) fraction IV: 3–10% A at 0–10 min, 10–15% A at 10–38 min, 15–25% A at 38–70 min, 25–40% A at 70–75 min, 40–95% A at 75–77 min.

C. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

37

Dry powder of H. diffusa (2.0 kg) Extracted by water and concentrated

An aliquot of crude extract (200 g) Dissolved in 1.2 L water-ethanol (20:80, v/v), kept static for 24 h and filtrated, respectively.

Filtrate: fraction A (26.7 g)

Water-soluble macro-components

Separated with XAD-4 macroporous resin column, eluted with water, 10 % ethanol, 30 % ethanol, 95 % ethanol (v/v)

Eluant of water: fraction B (6.6 g) Eluant of 10% ethanol: fraction C (8.8 g) Eluant of 30% ethanol: fraction D (4.3 g)

Effluents with strong UV absorbance: fraction I, fraction V-VIII

Eluant of 95% ethanol: fraction E (6.0 g) Separated by prep-RPLC with OEG column and eluted with water-methanol system by using gradient program.

Effluents with weak UV absorbance: fractions II, III, IV

Micro-components: analyzed by UPLC/Q-TOF MS Fig. 1. The procedure for preparation and isolation of micro-components.

3. Results and discussion 3.1. Enrichment and analysis strategy for micro-components from the extract of H. diffusa The objective of this work was to develop an efficient method for enrichment and analysis of micro-components in extracts of H. diffusa. Since the micro-components are often co-eluted with major components and the signals of micro-components are submerged in those of high-abundance counterparts, great efforts are required to solve the encountering difficulties during the process of isolation and detection of micro-components. In the work, efficient isolation method was applied through the following two steps: (1) Extracts of H. diffusa were fractioned by using the pretreatment method, which would reduce the complexity of the sample. (2) 2D-RPLC/RPLC system based on two dimensions with different separation mechanisms was developed aiming at enhancing the peak capacity of system, which would reduce the co-elution of micro-components from their counterparts. To reduce the complexity of the crude decocted extract of H. diffusa, pretreatment method was developed by using XAD-4 macroporous resin as the separation materials. With the pretreatment method, fractions could be isolated according to the polarity difference. The HPLC chromatograms of fractions B–E collected from the XAD-4 column and the extract by water extraction–alcohol precipitation (fraction A) were shown in Fig. 2a. The complexity of the four fractions B–E (Fig. 2b–e) was significantly reduced compared with that of fraction A (Fig. 2a). Additionally, fraction E was relatively more complicated than the other fractions (B–D) with the single-dimensional separation by using C18

stationary phase. Although only about 20 major peaks were observed in the spectrum of fraction E, micro-components might be co-eluted with the macro-components. To prove our assumption, fraction E was selected as sample for further isolation. To further isolate fraction E, 2D-RPLC/RPLC method was developed with Click OEG and C18 stationary phases as the first and second dimensions, respectively. Fraction E was primarily separated by using the Click OEG stationary phase with prep-RPLC. The preparative chromatogram of fraction E was shown in Fig. 3. Eight sub-fractions were obtained. It can be seen that the UV absorbance of these sub-fractions II, III and IV was approaching to the baseline. Two possible reasons contributed to the result: (1) The components in the three fractions showed with low content. (2) The components were weakly absorbed with UV detection under the detection wavelength. For better isolating and characterizing these sub-fractions, the efficient UV or MS detections as well as the 2D-LC system with high orthogonality should be considered. So UPLC/Q-TOF MS by using the C18 column was performed to isolate and characterize the fractions (II, III, IV) isolated by Click OEG stationary phase (Fig. 4). UPLC–diode array detector (UPLC–DAD) chromatograms at wavelength of 254 nm are listed in Fig. 4a, c, and e and UPLC/Q-TOF MS total ion chromatograms (in negative ion mode) in Fig. 4b, d, and f of fractions II, III, IV, respectively. It was found that majority of the peaks in the UPLC–DAD analysis had the relatively strong UV spectra, which indicated the contents of these components in fractions II, III, and IV were very low. With the secondary UPLC–DAD/Q-TOF MS analysis, more than 150 micro-components were observed in each sub-fraction, which were co-eluted with the major constituents. Among these detected micro-components, 38 compounds could be identified or

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mAU 175

a

150 125 100 75 50 25 0 mAU 250

b

200 150 100 50 0 mAU

c

1500 1250 1000 750 500 250 0 mAU

d

500 400 300 200 100 0

mAU 120

e

100 80 60 40 20 0 0

10

20

30

40

min

Fig. 2. The chromatograms of the extract by water extraction–alcohol precipitation (fraction A, Fig. 2a); the H2 O elution extract (fraction B, Fig. 2b), the 10% ethanol–water (v/v) elution extract (fraction C, Fig. 2c), the 30% ethanol–water (v/v) elution extract (fraction D, Fig. 2d) and the 95% ethanol–water (v/v) elution extract (fraction E, Fig. 2e) from XAD-4 porous resin of Hedyotis diffusa.

tentatively identified from fractions II, III, and IV. Therefore, it can be concluded that the micro-components were effectively isolated and characterized by using the developed method. 1500

3.2. Characterization of micro-components by using UPLC–DAD/Q-TOF MS

1200 900

A large number of micro-components were effectively isolated from the extracts of H. diffusa by using the 2D-RPLC/RPLC system and analyzed by using UPLC–DAD/Q-TOF MS. A total of over 400 compounds were separated, except some compounds detected overlapped in the three micro-component fractions. These compounds can be mainly classified into 12 classes (classes (1)–(12)) based on their UV spectra (Fig. 5). But only three types (5),

600

I V

300 0.0

II

10.0

III

VI

VII

VIII

IV

20.0

30.0

40.0

50.0 min

Fig. 3. The preparative chromatogram of fraction E for micro-components by prepRPLC at wavelength of 254 nm.

C. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

39

Fig. 4. UPLC/DAD (254 nm) chromatograms (a, c, e) and UPLC/Q-TOF MS total ion chromatograms (b, d, f) of fractions II, III, IV detected in negative ion mode, respectively.

40

C. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

Fig. 5. The main UV spectra types of the micro-components.

(6) and (8) of the compounds were tentatively/definitely identified with the respect of their UV spectra and MS spectra as phenylpropanoids, iridoid glucosides (IGs), and flavonoids without B-ring hydroxylation, respectively. Through the efficient separation with 2D-RPLC/RPLC system and structure characterization with UPLC–DAD/Q-TOF MS, a total of 38 compounds was identified or tentatively identified in the three fractions (II, III, IV), including 24 phenylpropanoids, 7 flavonoids, and 7 IGs. Most of the identified compounds have not previously been detected in H. diffusa. Table 1 showed the absorption maxima of the compounds and their identification on the basis of spectral data of the authentic standards and references in the literatures. The identification of these components was outlined below. 3.2.1. Characterization of phenylpropanoid micro-components The fragmentation pathways of phenylpropanoids are relatively characteristic in the negative ion mode with ESI source. The fragmentation pathways of phenylpropanoid were almost related to caffeoy acid, ferulic acid, coumaric acid, or quinic acid, which were confirmed by the typical ions [caffeoy acid–H]− at m/z 179, [ferulic acid–H]− at m/z 193, [coumaric acid–H]− at m/z 163, [quinic acid–H]− at m/z 191, or [quinic acid–H–H2 O]− at m/z 173 in the LC/MS analysis, respectively [13–16]. Thus, according to the typical UV and mass spectra characterization, 24 phenylpropanoids (peaks 1–4, 6–18, 25–31) were tentatively identified. The spectral information and identification of the 24 compounds are listed in Table 1. 3.2.2. Characterization of flavonoid micro-components Flavonoids as a kind of polyphenolic natural products also exist in the extract of H. diffusa. In the previous reports, the flavonoids in

H. diffusa were mainly quercetin, kaempferol, and their derivatives [17]. In the present work, seven compounds presented as peaks 19, 20, 32–34, 37, 38 in UPLC chromatogram of the three fractions (II, III, IV) were primarily identified as flavonoids according to their on-line UV spectra. The MS/MS spectra of [M−H]− (peaks 32–34, 38) or [M+H]+ (peak 37) are listed in Fig. 6. Their structural characterization was performed mainly based on their MS fragmentation behaviors (Table 1). By comparing the MS and UV spectra with the authentic standards, peaks 19 and 20 were identified as chrysin-6-C-ara-8-C-glu and chrysin-6-C-glu-8-C-ara, respectively. Peaks 19 and 20 were determined as isomers, exhibited [M−H]− ions at m/z 547. In their MS/MS spectra of the [M−H]− ions, peaks 19 and 20 could be confirmed as flavonoid C-glucosides due to the presence of [M−H−90]− ion at m/z 457 and [M−H−120]− ion at m/z 427. Noticeably, the two flavonoids were reported for the first time from the extracts in H. diffusa. Peaks 32 and 38 were also found as isomers, displaying the deprotonated molecule [M−H]− at m/z 459. Their mass spectra were similar, except for the abundance differences of the respective fragment ions derived from their ions of [M−H]− . Moreover, the two compounds both produced the ion of [M−H−176]− at m/z 283 as the base peak, which were originated from the neutral loss of a glucuronic acid residue. The observed fragment of m/z 268 proved the presence of a methoxyl group (m/z 283 → m/z 268), which indicated that peak 32 and 38 were methoxylated flavone. Thus peaks 32 and 38 were proposed as oroxylin A-O-glu acid and wogonin-O-glucuronide by comparing with data in literatures [18,19], respectively. Noticeably, peaks 32 and 38 were also found in this plant for the first time.

Table 1 Characterization of compounds from fractions II, III, IV. Peak

Retention time (min)

UV max (nm)

ESI− (m/z)

Elemental composition of [M−H]−

Error (ppm)

MS/MS of [M−H]− (m/z) (% base peak)

Identification

3-Caffeoylquinic acid [13,14] Methoxy-cinnamoyl hexoside p-Coumaric acid-O-glucoside Ferulic acid diglucoside [14]

[M−H]− 9.26 9.88 10.36 10.80

242, 303sh, 324 225, 277, 313 224, 294 236, 294sh, 325

353.0880 339.0724 325.0945 517.1533

C16 H17 O9 C15 H15 O9 C15 H17 O8 C22 H29 O14

2.0 2.4 6.8 −4.6

5a 6 7 8 9 10 11 12 13

10.99 11.23 12.06 13.00 13.50 14.02 14.72 15.08 15.91

237 239, 289sh, 318 231, 302sh, 309 230, 289sh, 308 238, 303sh, 324 245, 305sh, 326 239, 303sh, 325 244, 303sh, 325 237, 305sh, 323

413.1125 341.0862 337.0937 517.1544 367.1010 353.0850 517.1541 353.0852 517.1603

C18 H21 O11 C15 H17 O9 C16 H17 O8 C22 H29 O14 C17 H19 O9 C16 H17 O9 C22 H29 O14 C16 H17 O9 C22 H29 O14

−2.4 −3.2 4.2 −2.5 −5.2 −2.3 −3.1 −1.6 8.9

14

16.27

233, 305sh, 324

517.1561

C22 H29 O14

0.8

15 16 17 18 19b 20b 21a

17.88 19.42 21.11 21.84 27.34 30.47 36.70

229, 312 237, 303sh, 324 229, 311 238, 301sh, 325 220, 272, 313 225, 273, 313 236

337.0902 367.1011 337.0921 367.1011 547.1400 547.1413 493.1304

C16 H17 O8 C17 H19 O9 C16 H17 O8 C17 H19 O9 C26 H27 O13 C26 H27 O13 C23 H25 O12

−6.2 −4.9 −0.6 −4.9 −9.5 −7.1 −8.5

22a

42.18

240, 301sh, 314

549.1575

C26 H29 O13

−6.0

23a

42.89

240, 301sh, 314

549.1588

C26 H29 O13

−3.6

a

47.14

234

609.1705

C28 H33 O15

−9.2

25 26 27 28

8.84 9.92 11.56 13.32

229, 280 231, 280 244, 302sh, 326 236, 305sh, 324

325.0941 325.0915 487.1442 355.1034

C15 H17 O8 C15 H17 O8 C21 H27 O13 C16 H19 O9

5.5 −2.5 −2.1 1.4

29 30 31 32b 33b 34b

13.94 15.04 30.55 54.47 39.22 46.44

237, 322 237, 303sh, 324 233, 273 239, 273, 306 257, 353 238, 332

385.1157 355.1030 519.1727 459.0906 609.1486 831.1890

C17 H21 O10 C16 H19 O9 C22 H31 O14 C22 H19 O11 C27 H29 O16 C25 H29 O11

5.7 0.3 2.5 −4.6 4.9 14.1

135 (35), 179 (64), 191 (100) 133 (17), 177 (100) 119 (96), 163 (100) 71 (4), 101 (3), 113 (21), 134 (7), 175 (78), 193 (96), 217 (20), 235 (43), 265 (18), 337 (8), 355 (22), 397 (100) 101 (2), 119 (4), 147 (100), 191 (23), 251 (8) 135 (40), 179 (100) 119 (33), 163 (100), 191 (12) 193 (100), 355 (79), 401 (18), 499 (19) 134(41), 193 (100) 135 (37), 173 (100), 179 (60), 191 (49) 175 (38), 193 (32), 235(50), 265 (18), 295 (28), 355 (40), 397 (100) 191 (100) 175 (36), 193 (58), 235 (90), 265 (29), 295 (56), 355 (50), 397 (100), 427 (10), 134 (38), 149 (29), 175 (68), 191 (52), 193 (100), 235 (72), 265 (30), 295 (60), 355 (51), 397 (100), 427 (8), 457 (3), 473 (1) 119 (10), 163 (21), 173 (100) 134 (12), 173 (100), 193 (20) 93 (12), 163 (11), 191 (100) 134 (5), 173 (10), 191 (100) 281 (4), 309 (8), 337 (100), 367 (84), 427 (12), 457 (19), 487 (14), 529 (3) 281 (5), 309 (8), 337 (100), 367 (65), 427 (61), 457 (37), 487 (2), 529 (3) 101 (13), 121 (100), 147 (22), 165 (20), 179 (2), 191 (10), 251 (1), 269 (4), 287 (23), 313 (18), 331 (10), 371 (1) 101 (4), 163 (2), 227 (5), 255 (15), 285 (32), 293 (13), 337 (20), 369 (7), 473 (10), 517 (28), 549 (100) 101 (10), 163 (100), 241 (5), 285 (7), 293 (5), 337 (4), 369 (5), 473 (3), 517 (5) 101 (5), 121 (20), 131 (12), 149 (26), 193 (13), 271 (7), 315 (5), 487 (7), 473 (10), 609 (100) 113 (22), 119 (27), 145 (100), 163 (27), 187 (83), 217 (9), 265 (5) 119 (42), 145 (100), 163 (80), 187 (18), 191 (15), 281 (8) 135 (13), 161 (7), 179 (100), 233 (16), 251 (60), 323 (30) 134 (35), 149 (23), 160 (25), 175 (68), 193 (100), 235 (75), 265 (38), 295 (62) 164 (33), 190 (32), 205 (57), 223 (100), 265 (91), 295 (50), 325 (68) 134 (33), 149 (22), 175 (63), 193 (100), 235 (89), 265 (29), 295 (65) 136 (2), 151 (41), 342 (2), 357 (100) 113 (40), 175 (12), 268 (70), 283 (100) 179 (2), 227 (2), 255 (6), 284 (100), 301 (90) 151 (1), 179 (2), 300 (21), 463 (1), 625 (33), 831 (100)

35a

51.78

233, 312

551.1671

C26 H31 O13

17.1

101 (37), 163 (100), 287 (12), 357 (20), 389 (21)

36a 37b

54.57 56.51

234 228, 256, 352

477.1327 955.3030

C23 H25 O11 C49 H47 O20

−14.7 18.6

38b

59.53

230, 272, 311

459.0901

C22 H19 O11

−5.7

24

101 (15), 121 (100), 149 (49), 175 (30), 193 (16), 271 (5), 355 (3) (+)127 (1), 169 (9), 229 (5), 295 (12), 303 (100), 331 (85), 465 (15), 493 (45), 655 (2) 113 (24), 239 (2), 268 (100), 283 (75)

Asperulosidesd Caffeoyl hexoside [14] 3-р-Coumaroylquinic acid [13] Ferulic acid diglucoside[14] 3-Feruloylquinic acid [13,14] 4-Caffeoylquinic acid [13,14] Ferulic acid derivative [14] 5-Caffeoylquinic acid [13,14] Ferulic acid derivative [14] Ferulic acid derivative [14] 4-р-Coumaroylquinic acid [13] 4-Feruloylquinic acid [13,14] 5-р-Coumaroylquinic acid [13,14] 5-Feruloylquinic acid [13,14] Chrysin-6-C-ara-8-C-Glcsd Chrysin-6-C-glc-8-C-Arasd 10-O-Benzoyl-sandoside or 10-O-benzoyl-deacetyl-asperuloside acid Z-6-O-p-Coumaroyl scandoside methyl ester isomer E-6-O-p-Coumaroyl scandoside methyl ester isomer Oldenlandoside III Coumaric acid-O-glucoside [16] Coumaric acid-O-glucoside [16] Caffeic acid derivative Ferulic acid hexoside [14] [15] Sinapoyl glucose [15] Ferulic acid hexoside [14,15] Pinoresinol-O-glucopyranoside [18] Oroxylin-A-O-glu acid [17,18] Rutinsd Quercetin-3-O-[2- (6-O-E-sinapoyl-␤-Dglucopyranosyl-␤-D-glucopyranosyl [20] E-6-O-p-Coumaroyl dihydroscandoside methyl ester isomer Hehycoryside C [24] Quercetin derivative

C. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

1 2 3 4

Wogonin-O-glu acid [17,18]

a

41

Iridoid glycoside. b Flavonoid: the other: phenylpropanoid. sd : The MS data were identical with those of corresponding standard.

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283

Peak32

268 113

%

100

75 85 99 114 129

0 50

459

150

200

284 301

Peak33

%

100

100

284

175

125 164 179 227255

0 50

100

150

200

283

300

300

400

350

400

450

500

550

600

%

300

151 179

100

271

299 302

200

400

650

831

500

600

700

750

700

800

900

% 0

100

200

303 281

300

400

Peak38

500

268

m/z 1000 957 958

493 498 499 366 489 547

365

655 709 769 795 848 901

600

700

800

900

979 982

m/z 1000

283

%

100

158 214

m/z 800

833

100 Peak37 117 114 70

m/z 550 723

500

832

625 626 607

445

300

609

450

608 611

429

301

0

350

610

302 325

250

100 Peak34

250

0 50

71

85

113 99 117 163 175 198

100

150

200

239

267

284

250

300

459

350

400

450

500

m/z 550

Fig. 6. The MS/MS spectra of [M−H]− ions of peaks 32–34, 38 and MS/MS spectra of [M+H]+ ion of peak 37.

The ions observed in MS/MS spectra of peaks 33, 34 and 37 are the typical ones for the fragmentation of quercetin derivatives, displaying the ion [quercetin+H]+ at m/z 303 in positive mode and the ion [quercetin−H]− at m/z 300/301. The major fragment ions at m/z 179, 151 of the [quercetin−H]− ion were almost observed. Peak 33 was identified as rutin through comparison with the authentic standard, which has not been detected from H. diffusa. Peak 34 was tentatively identified as quercetin-3-O-[2-(6-O-E-sinapoyl␤-d-glucopyranosyl-␤-d-glucopyranosyl, which has been isolated from H. diffusa [20]. Distinguishingly, the positive ion MS/MS spectrum of [M+H]+ of peak 37 was employed, which is due to the weak fragment ions of its [M−H]− in the applied experimental MS conditions. And the fragment ion of [quercetin+H]+ at m/z 303 of [M+H]+ of peak 37 was obviously observed. 3.2.3. Characterization of IG micro-components IGs as one of the main classes of components in H. diffusa, have the characteristic UV spectra with the maximum absorbance at the wavelength of 230–240 nm. Seven IGs (peaks 5, 21–24, 35, 36) were detected in the micro-component fractions on the basis of their characteristic UV and MS information (Table 1). Their MS spectra are listed in Fig. 7. Among them, compound presented as peak 24 has the same molecular weight (MW) of 610 Da as oldenlandoside III, which has been separated from H. duffusa [21]. In the MS/MS spectra of [M−H]− ion, the fragment ion at m/z 487 was produced by the neutral loss of a molecule benzoic acid, corresponding to [M−H−122]− ion. Fragment ion at m/z 315 was associated with the losses of a

glycosyl (Glc) unit (−162 Da) and an ara unit (−132 Da). The ion at m/z 121 can be regarded as the deprotonated ion of benzoic acid. The ion at m/z 101 was corresponded to the characteristic fragment ion 2,7 F0 − of 7,8-cyclopentene-type IG [12]. Thus, peak 24 was primarily identified as oldenlandoside III. Peak 21 yielded an ion of [M−H]− at m/z 493.1304 (C23 H25 O12 , calculated by its accurate molecular mass). In the MS/MS spectrum of [M−H]− , the fragment ion at m/z 371 was also yielded by the loss of a molecule benzoic acid as that of peak 24, which was further confirmed through the base peak ion of [benzoic acid−H]− at m/z 121. Similarly, The ion at m/z 101 corresponded to the characteristic fragment 2,7 F0 − ion of 7,8-cyclopentene-type IG [12]. Noticeably, an ara unit (132 Da) was less and an OH group (17 Da) was more of peak 21 than that of peak 24, which was also consistent with the fragment ions. Due to the special position of OH group at C-6 position, peak 21 was tentatively identified as 10-O-benzoyl-scandoside or 10-O-benzoyl-deacetylasperuloside acid. To the best of our knowledge, this structure has not been reported from the extracts of H. diffusa. But the similar structure of 10-O-benzoyl-scandoside methyl ester has been isolated from H. diffusa [21] and of 10-Obenzoyl-deacetylasperuloside acid methyl ester has been isolated from the congeneric plant, Oldenlandia corymbosal [22]. As shown in Table 1, peaks 22 and 23 corresponded to the isomers with MWs of 550 Da. The [M−H]− ions of peak 22 and peak 23 both were selected as the precursor ions in the MS/MS experiment to give respective fragmentation information (Fig. 7). It was found that the fragment ions in MS/MS spectrum of peak 22 were very similar to those of peak 23, except for their intensity. Taking

C. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

147

Peak 5

100

- CO2

[M-H] 413

%

-CH3COOH 101

0 50

100 %

Peak 21

191 179

150

- Glc 327

233 251

200

250

300

350

-CO2 147 165 179

150

269

191

200

314

287

250

300

%

100

150

255

227

200

285

250

[coumaroyl acid-H] 163

293 337

300

101 119 162 164

100

450

500

150

200

m/z

400

450

550

600

650

700

600

650

700

m/z

549

250

- 180 550

300

350

400

450

551

517

473

500

550

m/z

-122 -Glc-Ara

193

150

Peak 35

500

609

121 149 101

100

550 517 518 551

473

% 100

600

494

-CH3OH

241 285 223 293 337 369

-CO2

0 50

550

m/z

-

Peak 24

71 89

600

495

400

369

350

% 100

550

549

149

71

-122

371

350

-CH3OH

0 50

500 493

-Glc

313

-Glc-102

71 101

Peak 23

450

-Glucose

Peak 22

100

400

-122

100

0 50

[M+HCOO] 414 459

121

101

0 50 100

148

119

100

71 59

43

253 271

200

250

487

315

300

350

- coumaric acid

[coumaric acid-H] 163

400

450

610

-122 (-benzoic acid)

500

550

611

600

650

700

m/z

- 102 %

101 119 145

0 50

100

150

200

[benzoyl acid-H]121

250

0 50

71

150

357

350

389

400

551 552

- Glc 450

500

550

600

m/z

- Glc - CO2

175 193 253 150 271 209

101

100

329

300

131149 59

287

- 122

Peak 36

%

100

69

-CH3OH 164 193 225 243

200

250

300

315

350

-122

355

400

450

477

500

550

m/z

Fig. 7. The MS/MS spectra of [M−H]− ions of peaks 5 and 21–24, 35, 36.

account of the UV spectra with maximum absorbance around the wavelength of 320–330 nm and a shoulder around 300–310 nm, peaks 22 and 23 maybe contain phenylpropanoid substituents, which was demonstrated by the detection of product ion at m/z 163, corresponding to a coumaric acid group. A neutral loss of a glc unit (m = 162 Da) was often observed in the MS/MS spectra of IGs, the Y− 0 ion [12] for the two compounds appearing at m/z 387 with a very low intensity. The product ion at m/z 517, formed by the

neutral loss of CH3 OH, indicated the presence of a methyl ester group at the C-4 position. The ion at m/z 473 was the result of successive losses of the CH3 OH and CO2 from the [M−H]− ion at m/z 549. The diagnostic product ion 2,7 F− 0 at m/z 101 of 7,8cyclopentene-type IGs [12] was also observed in the MS/MS spectra of peak 22 and 23. Based on the fragmentation, peak 22 and 23 were tentatively identified as Z/E-6-O-p-coumaroyl scandoside methyl ester, which have been isolated from H. diffusa [17]. It is also worthy

44

C. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 99 (2014) 35–44

of note that the product ion at m/z 163 was the base peak for peak 23, but only with 3% intensity of base peak for peak 22, which probably arose from the stereochemical difference of the C-6 coumaroyl group in peak 22 and 23. Therefore, it was deduced that the C-6 coumaroyl group in peak 23 may be above the plane like the C-1 substituent [23]. Thus, peak 23 was tentatively identified as E-6-Op-coumaroyl scandoside methyl ester, and peak 22 probably was Z-6-O-p-coumaroyl scandoside methyl ester. Peak 35 was identified as IG, based on its MS/MS spectrum of [M−H]− at m/z 551 and UV spectrum. According to the base peak at m/z 163 and the UV spectral behavior, peak 38 was also identified as IG containing a coumaric acid moiety. Neutral losses of a glc unit and of successive coumaric acid were observed at m/z 389 and at m/z 225, respectively. The fragment ion at m/z 287 was produced by the loss of 102 Da from the aglycone moiety ion of m/z 389, and the intensity of the corresponding ion of m/z 101 was relatively strong. Thus, the ion at m/z 101 with relatively high abundance was confirmed as the characteristic ion 2,7 F0 − of cyclopentane-type IG [12]. Additionally, the neutral loss of CH3 OH (m/z 389 → m/z 357) in the MS/MS spectra of the [M−H]− ion indicated the presence of a methyl ester group at the C-4 position of the compound responsible for peak 38. Based on the fragmentation behavior, the proposed structure of peak 35 was similar to that of peak 23. The only difference between the two compounds was the latter was considered as cyclopentene-type IG, but not a cyclopentane-type IG as that of the former. Furthermore, the MW of peak 35 was just 2 Da more than that of peak 23. According to the proposed structure, peak 35 was tentatively identified as E-6-O-p-coumaroyl dihydroscandoside methyl ester, which has not been reported from extract of H. diffusa in literatures. The MW of peak 36 was 478 Da, which was confirmed by the [M−H]− ion at m/z 477.1327 with the formula of C26 H31 O13 . The [M−H]− ion of peak 36 was 132 Da less than that of peak 24, which can be accounted for an ara unit. Therefore, peak 36 could be proposed as hehycoryside C, which has been isolated from the congeneric plant, H. corymbosa (L.) Lam [24], but has not been observed from H. diffusa. 4. Conclusions A large number of micro-components in H. diffusa were effectively enriched and isolated with the 2D-RPLC/RPLC system by using the Click OEG stationary phase as the first dimension and C18 stationary phase as the second dimension, and the components were characterized by using UPLC–DAD/Q-TOF MS. More than 400 micro-components were isolated and detected with the developed method. Among these components, 38 micro-components, involving 24 phenylpropanoids, 7 flavonoids and 7 IGs, were identified or tentatively identified in the extract of H. diffusa on the basis of spectral data of the authentic standards and the fragmentation characteristic information available in literatures. Among the detected micro-components, majority of them have not been observed in H. diffusa. Acknowledgments The authors gratefully acknowledge the financial support from the Key Projects in the National Science & Technology Pillar Program in the twelfth Five-year Plan (No. 2012BAI29B08), the National Science Foundation of China (No. 21105007), and the Natural Science Foundation of Hebei Province of China (No. B2013208018).

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