Characterization and identification of the chemical constituents in the root of Lindera reflexa Hemsl. using ultra-high performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry

Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

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Characterization and identification of the chemical constituents in the root of Lindera reflexa Hemsl. using ultra-high performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry Xiaoya Sun a,c , Yunbin Zhang b , Suiqing Chen a,c,∗ , Yu Fu a a

School of Pharmacy, Henan University of Traditional Chinese Medicine, Boxue Road, Jinshui District, Zhengzhou 450046, China Henan Province Hospital of Traditional Chinese Medicine, Dongfeng Road, Jinshui District, Zhengzhou 450002, China c Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, Boxue Road, Jinshui District, Zhengzhou 450046, China b

a r t i c l e

i n f o

Article history: Received 1 February 2016 Received in revised form 12 April 2016 Accepted 17 April 2016 Available online 21 April 2016 Keywords: Lindera reflexa Hemsl. UHPLC-LTQ-Orbitrap-MS Identification Diagnostic fragmentation

a b s t r a c t The root of Lindera reflexa Hemsl. (LR) is a newly discovered herbal drug and has been used to treat gastritis and peptic ulcers. Nevertheless, the chemical profile of LR has not been established. In this study, ultra-high performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS) was performed to investigate the fragmentation behaviors of multiple compounds from LR. The 12 standards were divided into five types according to their basic skeletons: stilbenes, flavonoids, alkaloids, pyranone I and pyranone II. The MSn spectra of the [M+H]+ or [M±NH4 ]+ ions for these compounds provided a wealth of structural information on the five different types of compounds. Stilbenes yielded ions with successive loss of 78 Da (C6 H6 ) and 28 Da (CO). The subsequent loss of H2 O, CO, RDA and C-ring fragmentation were the most possible fragmentation pathways for flavonoids. Fragmentation with successive loss of 17 Da (NH3 ) or 31 Da (CH5 N), 32 Da (CH4 O) and 28 Da (CO) in the MSn spectra were characteristics of alkaloids. The characteristic ions for Pyranone I were m/z 255.1013, m/z 243.1013 and m/z 237.0909, and the diagnostic ions for Pyranone II were m/z 227.0700, m/z 215.0700, m/z 185.0594 and m/z 131.0489. Using accurate mass measurements for each precursor ion and the subsequent fragmented ions, a total of 42 compounds were identified or tentatively characterized in LR, including 24 potentially new compounds. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Lindera reflexa Hemsl. (LR) is a type of shrub or vine in the family of Lauraceae that is mainly distributed in the southern region of the Yangtze River in China. LR root has been listed in the Dictionary of Chinese Medicine and prescribed for the treatment of gastritis and peptic ulcers in the past few years [1]. Various chemical constituents, including volatile oils [2], stilbenes [3–7], flavonoids [4], alkaloids [8–11], pyranone I [1] of reflexan A and pyranone II [12,13] of katsumadain, have been isolated from LR root. Among these compounds, our laboratory discovered reflexan A, reflexanbene I and reflexanbene II [1].

∗ Corresponding author at: School of Pharmacy, Henan University of Traditional Chinese Medicine, Boxue Road, Jinshui District, Zhengzhou 450046, China. E-mail address: [email protected] (S. Chen). http://dx.doi.org/10.1016/j.jpba.2016.04.023 0731-7085/© 2016 Elsevier B.V. All rights reserved.

Moreover, reflexan A possesses a novel skeleton. Their antitumor activities and anti-inflammatory potencies have been investigated in various systems in our previous study. Additionally, stilbenes possessed many biological activities, including antibacterial, antifungal [14], cancer chemopreventive, anti-oxidative stress, and anti-inflammation activities [15–17]. Alkaloids exhibit significant biological activities, including antiplatelet aggregation, adrenoceptor antagonism, antiserotonergic activity, ion channel inhibition, antioxidative activity, effects on the nervous system, immunomodulatory activity, antivirus activity, etc. [18–20]. The 2-pyranone derivatives display antitumor, antimicrobial, and anti-HIV activities [21]. However, to the best of our knowledge, there were only a few papers concerned with the analysis of these compounds in LR root, and these studies only focused on the main compounds. In this study, a highly sensitive and rapid analytical method was employed for the systematic analysis of the multiple constituents of LR.

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

With the rapid development of chromatography and mass spectrometry (MS) techniques, ultra-high performance liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap mass spectrometry (UHPLC-LTQOrbitrap-MS) has become an extremely powerful tool for the identification of natural products from complex matrices, including flavonoids, phenolic acids, glycosides, and alkaloids in medicinal plants [22–24]. Compared with the low resolution mass spectrometry methods, LTQ-Orbitrap-MS not only provides high sensitivity with a mass accuracy of less than 5 ppm but also offers an unequivocal assignment of the product ions to their respective precursor ions in MSn (n ≥ 3) experiments, allowing us to precisely describe the fragmentation pathways. In this paper, we focused on a comprehensive study of the MSn spectra on a LTQ-Orbitrap-MS to elucidate the fragmentation behavior of 12 known compounds isolated from LR root and also applied the characteristic fragmentation patterns to preliminarily identify additional unknown chemical constituents from this herbal medicine. In this study, 12 compounds were divided into five types according to their basic skeletons: stilbenes, flavonoids, alkaloids, pyranone I and pyranone II. Based on the fragmentation patterns, accurate mass measurement and retention time, a total of 42 compounds were unambiguously identified or tentatively characterized, 24 of which are potentially new compounds. This is the first determination of multiple components in LR root using UHPLC-LTQ-Orbitrap-MS.

35

macroporous resin column, and the 95% aqueous methanol elution fraction was concentrated. The dried residue was redissolved in 50 mL of methanol in a volumetric flask. All of the samples were filtered through a 0.22-␮m membrane and directly analyzed using UHPLC-LTQ-Orbitrap-MS.

2.4. LC conditions The chromatographic analysis was performed on a Dionex UltiMate 3000 UHPLC system (Thermo Scientific, Germering, Bavaria, Germany) equipped with a binary pump, an online degasser, a thermostatted autosampler, a thermostatically controlled column compartment, and a diode array detector (DAD). Chromatographic separation was performed on a reverse-phase column Syncronis C18 column (2.1 × 100 mm, 1.7 ␮m, Thermo Scientific) maintained at 30 ◦ C.The mobile phases consisted of water containing 0.1% formic acid (A) and acetonitrile (B), and the elution gradient was set as follows: 0.0–1.0 min, 15.0% B; 1.0–28.0 min, 15.0–100.0% B; 28.0–31.0 min, 100% B; 31.0–31.1 min, 100.0–15% B; and 31.1–33.0 min, 15.0% B. The flow rate was set at 0.3 mL/min, and the injection volume was 1 ␮L.

2. Materials and methods

2.5. Mass spectrometry and data processing

2.1. Plant material

For the LC-ESI-MSn experiments, a Thermo Fisher LTQ-Orbitrap XL Hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an electrospray ionization (ESI) source was connected to the UHPLC instrument. The ESI source parameters were set as follows: ion spray voltage, 4.5 kV; capillary temperature, 350 ◦ C; capillary voltage, 15 V; tube lens voltage, 80 V; and sheath (N2 ) and auxiliary gas (He) flow rates, 25 and 3 arbitrary units, respectively. The Orbitrap mass analyzer was operated in positive ion mode, with a mass range of 80–2000. Tuning methods were developed using a multi-objective optimization experiment, similar to the technique described for ESI-MS. Accurate masses were calibrated according to the manufacturer’s guidelines using a standard mixture of caffeine, MRFA and Ultramark 1621. The Fourier transform resolutions were set at 30,000 (full width at half maximum, as defined at m/z 400) for MS and MSn (n = 4). The MS and MSn data were recorded in the profile and centroid formats, respectively. The average acquisition time required to finish a scan cycle (containing four scan events) was 3.6 s. The most intense ions detected in the full-scan spectrum were selected for the data-dependent scan. The normalized collision energy for collision-induced dissociation (CID) was adjusted to 30% of the maximum, the isolation width of the precursor ions was m/z 2.0, and the default values were used for the other CID parameters. To simultaneously obtain the information about the fragments for co-eluting minor peaks, dynamic exclusion was enabled using the following optimal parameters: repeat count, 2; repeat duration, 10 s; exclusion duration, 10 s. A series of potential interfering or contaminating ions were added to the exclusion list to avoid acquiring their MS n data. The data were recorded and processed using the Xcalibur 3.0 software (Thermo Fisher Scientific) and Mass Frontier 7.0 software (Thermo Fisher Scientific, Waltham, MA, USA). Considering the possible elemental compositions of the potential components present in the LR samples, the elements in use (C 0–50, H 0–100, O 0–50, N 0–5), ring double-bond equivalent (RDB equivalent value −1.0–100.0), and mass tolerance (<5 ppm) were set to reduce the number of options used to determine the elemental compositions of both the precursor and product ions.

The LR roots were collected from Xinyang, Henan, China (GPS coordinates 31◦ 34 28.1 N 114◦ 51 57.4 E), in March 2015 and authenticated by one of the authors, Prof. Suiqing Chen (Department of Pharmacognosy, Henan University of Traditional Chinese Medicine) according to the morphological characteristics. A voucher specimen (voucher number 20150318) was deposited in the Pharmacognosy Lab of the Pharmacy College, Henan University of Traditional Chinese Medicine, Henan, China. 2.2. Chemicals and reagents The standards, including the stilbenes of pinosylvin, 3methoxy-5-hydroxy-trans-stilbene, ␤,␤ -pinosylvin diglucoside, reflexanbene I, reflexanbene II; flavonoids of pinostrobin and pinocembrin; alkaloids of launobine, laetanine, norbracteoline; katsumadain; and reflexan A were isolated from the LR in our laboratory [1]. Their structures (Fig. 1) were unequivocally elucidated by spectroscopic methods (IR, MS, 1 H NMR and 13 C NMR). The purity of the standards was determined to be higher than 98% by normalizing the peak area using HPLC-DAD (diode array detector). Stock solutions (0.1 mg/ml) of the 12 standards were prepared individually in methanol. Acetonitrile (ACN) and formic acid (both HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Methanol (HPLC grade) was purchased from Merck (Darmstadt, Germany). Deionized water was prepared by passing distilled water through a Milli-Q water purification system (Millipore, Milford, MA, USA). 2.3. Sample preparation The LR roots were pulverized into a powder (50.0 g, 24 mesh), weighed, soaked in 500 mL of water for 0.5 h, and distilled for 4 h by hydrodistillation. After filtration, the residue was placed in a stoppered conical flask containing 600 mL of 70% ethanol and extracted by ultrasonication for 1 h. The above extraction procedures were repeated twice. The 70% ethanol extract was loaded on a DM130

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X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

1. Stilbenes

Compound β,β′-pinosylvin diglucoside (10)

R1 glucose

R2 glucose

R3 H

Pinosylvin (12) 3-methoxy-5-hydroxy-trans-stilbene (16)

H H

H CH3

H H

Reflexanbene I (33)

H

H

1-p-mentheneyl

Reflexanbene II (18)

H

H

1-(1,2-epoxy)-phmentheneyl

3. Alkaloids

2. Flavonoids

Compound Pinostrobin (19)

R CH3

Compound

R1

R2

Laetanine (3)

OH

OCH3

Pinocembrin (13)

H

Norbracteoline (1)

OCH3

OH

4. Pyranone Ι

Launobine(6)

5. Pyranone ΙΙ

Reflexan A (26)

Katsumadain (27)

Fig. 1. Chemical structures of the five different types of compounds identified from L. reflexa Hemsl.

3. Results and discussion 3.1. Optimization of the LC and MS conditions The UHPLC-LTQ-Orbitrap-MS conditions were systemically optimized to improve the resolution and sensitivity of the analysis and obtain appropriate ionization. The LC conditions, including the type of chromatographic columns and mobile phase systems, were investigated; the results indicated that the Syncronis C18 column with a small (1.7 ␮m) particle size provided better separation with a higher peak resolution and that aqueous acetonitrile with 0.1% formic acid was superior to the other mobile phases. The LTQ-Orbitrap conditions were also systematically optimized; accordingly, a resolution power of 30,000 in full scan mode, followed by MSn scan in the Orbitrap (at R = 30,000), a 10 s exclusion duration, and 30% of the maximum CID collision energy were employed to obtain appropriate ionization and MS abundance information in positive ion mode.

3.2. Identification of the main constituents of LR Based on the structural characteristics of the 12 reference compounds, they were divided into five groups: stilbenes, including pinosylvin (12), 3-methoxy-5-hydroxy-trans-stilbene (16), ␤,␤ pinosylvin diglucoside (10), reflexanbene I (33) and reflexanbene II (18); flavonoids, including pinocembrin (13) and pinostrobin (19); alkaloids, including launobine (6), laetanine (3) and norbracteoline (1); pyranone I, consisting of reflexan A (26); and pyranone II, consisting of katsumadain (27). The fragmentation

patterns and pathways of these reference compounds are shown in Table 1. Moreover, the fragmentation patterns and pathways of the standards were investigated in depth to further confirm the structures of their derivatives. For the unavailable standard compounds, the structures were presumed based on the following steps to increase the credibility: (1) the molecular formula was established based on high-accuracy protonated precursors such as [M+H]+ and [M±NH4 ]+ within a mass error of 5 ppm and the fractional isotope abundance; (2) when there were several matching isomers, the structures were identified in the chemical information database of the LR components; and (3) the fragment ions from MSn mass spectrometry were used to further confirm the chemical structure with the aid of Thermo Scientific TM Mass Frontier 7.0. In particular, for the flavonoids, pinostrobin, pinocembrin, etc., the fragmentation rules for the standards were applied to elucidate the structures of the corresponding derivatives with the same basic skeleton. The total ion chromatograms (TIC) of the LR samples were acquired in positive mode for further confirmation (shown in Fig. 2). Based on the MSn data, accurate mass measurement and retention time, a total of 42 components, including 8 stilbenes, 8 flavonoids, 9 alkaloids, 10 pyranone I compounds and 7 pyranone II compounds, were unambiguously or tentatively identified from LR, which are summarized in Table 2.

3.2.1. Stilbenes In the present study, 5 reference compounds, pinosylvin (compound 12), 3-methoxy-5-hydroxy-trans-stilbene (compound 16), ␤,␤ -pinosylvin diglucoside (compound 10), reflexanbene I (compound 33), and reflexanbene II (compound 18), were first

Table 1 MSn data and proposed fragmentation pathways of the 12 reference compounds. Type Stilbenes

Standard 

␤,␤ -pinosylvin diglucoside (10)

selection +

[M+NH4 ]

Molercular formula

Error (ppm)

MSn

Fragment ions

Elem.Comp.

Error (ppm)

Pathways

554.22247

C26 H36 O12 N

−1.321

MS2 [554.22]

537.19598

C26 H33 O12

−1.252

[M+NH4 −NH3 ]+

375.14331 213.09073 195.08017 167.08522 135.04384 107.04894 91.05414 107.04895 195.08047 167.08546 135.04402 107.04907 91.05415 107.04890 209.09595

C20 H23 O7 C14 H13 O2 C14 H11 O C13 H11 C8 H7 O2 C7 H7 O C7 H7 C7 H7 O C14 H11 O C13 H11 C8 H7 O2 C7 H7 O C7 H7 C7 H7 O C15 H13 O

−1.385 −1.296 −1.392 −1.837 −1.600 −1.881 −0.953 −1.788 0.146 −0.401 −0.267 −0.667 −0.844 −2.255 −0.677

[M+NH4 –NH3 −C6 H10 O5 ]+ [M+NH4 −NH3 −2C6 H10 O5 ]+ [M+NH4 −NH3 −2C6 H10 O5 −H2 O]+ [M+NH4 −NH3 −2C6 H10 O5 −H2 O−CO]+ [M+NH4 −NH3 −2C6 H10 O5 −C6 H6 ]+ [M+NH4 −NH3 −2C6 H10 O5 −C6 H6 −CO]+ [M+NH4 −NH3 −2C6 H10 O5 −C7 H6 O2 ]+ [M+NH4 −NH3 −2C6 H10 O5 −C6 H6 −CO] + [M+H−H2 O]+ [M+H−H2 O−CO]+ [M+H−C6 H6 ]+ [M±H−C6 H6 −CO] + [M+H−C7 H6 O2 ]+ [M+H−C6 H6 −CO] + [M+H−H2 O]+

195.08034 181.10110 167.08534 149.05959 121.06473 91.05412 121.06464 347.20004 291.13696 265.12210 225.09061 213.09073 213.09100 195.07999 167.08504 135.04375 1107.04887 293.15332 239.10658 225.09076 213.09073 137.13222 195.07990 167.08525

C14 H11 O C14 H13 C13 H11 C9 H9 O2 C8 H9 O C7 H7 C8 H9 O C24 H27 O2 C20 H19 O2 C18 H17 O2 C15 H13 O2 C14 H13 O2 C14 H13 O2 C14 H11 O C13 H11 C8 H7 O2 C7 H7 O C20 H21 O2 C16 H15 O2 C15 H13 O2 C14 H13 O2 C10 H17 C14 H11 O C13 H11

−0.520 −0.425 −1.119 −0.779 −0.508 −1.173 −0.151 −1.488 −3.422 −0.778 −1.760 −1.296 −0.029 −2.315 −2.914 −2.266 −2.535 −0.977 −0.319 −1.094 −1.296 −1.875 −0.542 −0.277

[M+H−CH4 O]+ [M+H−H2 O−CO]+ [M+H−CH4 O−CO]+ [M+H−C6 H6 ]+ [M+H−C6 H6 −CO]+ [M+H−C8 H8 O2 ]+ [M+H−C6 H6 −CO]+ [M+H−H2 O]+ [M+H−H2 O−C4 H8 ]+ [M+H−H2 O−C6 H10 ]+ [M+H−H2 O−C9 H14 ]+ [M+H−H2 O−C10 H14 ]+ [M+H−H2 O−C4 H8 −C6 H6 ]+ [M+H−H2 O−C10 H14 −H2 O]+ [M+H−H2 O−C10 H14 −H2 O−CO]+ [M+H−H2 O−C10 H14 −C6 H6 ]+ [M±H−H2 O−C10 H14 −C6 H6 −CO] + [M+H−C4 H8 ]+ [M+H−C8 H14 ]+ [M+H−C9 H16 ]+ [M+H−C10 H16 ]+ [M+H−C14 H12 O2 ]+ [M+H−H2 O]+ [M+H−H2 O−CO]+

MS3 [213.09]

Pinosylvin (12)

[M+H]+

213.09085

C14 H13 O2

−0.733

MS4 [135.04] MS2 [213.09]

3-methoxy-5-hydroxytrans-stilbene (16)

[M+H]+

227.10681

C15 H15 O2

0.677

MS3 [135.04] MS2 [227.11]

Reflexanbene II (18)

[M+H]+

365.21048

C24 H29 O3

−1.756

MS3 [149.05] MS2 [365.21] MS3 [347.20]

MS4 [291.14] MS4 [213.09]

Reflexanbene I (33)

[M+H]+

349.21622

C24 H29 O2

0.038

MS2 [349.22]

MS3 [213.09]

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

Precursor ions

37

38

Table 1 (Continued) Type

Alkaloids

selection

Precursor ions

Molercular formula

Error (ppm)

MSn

Pinocembrin (13)

[M+H]+

257.08072

C15 H13 O4

−0.449

MS2 [257.08]

Pinostrobin (19)

[M+H]+

271.09665

C16 H15 O4

0.607

MS2 [271.10]

Norbracteoline (1)

[M+H]+

314.13870

C18 H20 O4 N

−0.049

MS2 [314.14] MS3[ 297.11] MS4 [265.09]

Laetanine (3)

[M+H]+

314.13855

C18 H20 O4 N

−0.428

MS2 [314.14] MS3 [297.11] MS4 [265.09]

Fragment ions

Elem.Comp.

Error (ppm)

Pathways

135.04367 107.04881 239.06999 215.07008 179.03363 173.05945 153.01797 145.06458 131.04895 103.05405 253.08598 229.08598 193.04945 173.05959 167.03380 131.04909 103.05415 297.11188 282.08847 265.08572 250.06227 247.07507 237.09079 233.05986 222.06696 205.06465 297.11230 282.08887 265.08609 250.06256 237.09106 233.05986 222.06731

C8 H7 O2 C7 H7 O C15 H11 O3 C13 H11 O3 C9 H7 O4 C11 H9 O2 C7 H5 O4 C10 H9 O C9 H7 O C8 H7 C16 H13 O3 C14 H13 O3 C10 H9 O4 C11 H9 O2 C8 H7 O4 C9 H7 O C8 H7 C18 H17 O4 C17 H14 O4 C17 H13 O3 C16 H10 O3 C17 H11 O2 C16 H13 O2 C16 H9 O2 C15 H10 O2 C15 H9 O C18 H17 O4 C17 H14 O4 C17 H13 O3 C16 H10 O3 C16 H13 O2 C16 H9 O2 C15 H10 O2

−0.386 −0.331 −1.174 −0.887 −1.425 −1.480 −1.733 −1.458 −1.461 −1.716 0.234 0.258 −0.442 −0.671 −0.510 −0.392 −0.745 −0.860 −0.675 −0.757 −0.703 −1.158 −0.912 0.660 −2.572 −0.690 0.554 0.743 0.638 0.457 0.227 0.660 0.085

[M+H−C6 H6 ]+ [M±H−C6 H6 −CO] + [M+H−H2 O]+ [M+H−C2 H2 O]+ [M+H−C6 H6 ]+ [M+H−2C2 H2 O]+ [M+H−C8 H8 ]+ [M+H− 2C2 H2 O−CO]+ [M+H−3C2 H2 O]+ [M+H−3C2 H2 O−CO]+ [M+H−H2 O]+ [M+H−C2 H2 O]+ [M+H−C6 H6 ]+ [M+H−C2 H2 O−C3 H4 O]+ [M+H−C8 H8 ]+ [M+H−C7 H8 O3 ]+ [M+H−C7 H8 O3 −CO]+ [M+H−NH3 ]+ •+ [M+H−NH3 −CH3 ] [M+H−NH3 −CH4 O]+ •+ [M+H−NH3 −CH4 O−CH3 ] [M+H−NH3 −CH4 O−H2 O]+ [M+H−NH3 −CH4 O−CO]+ [M+H−NH3 −CH4 O−CH4 O]+ •+ [M+H−NH3 −CH4 O−CH3 −CO] [M+H−NH3 −CH4 O−2CH2 O]+ [M+H−NH3 ]+ •+ [M+H−NH3 −CH3 ] [M+H−NH3 −CH4 O]+ •+ [M+H−NH3 −CH4 O−CH3 ] [M+H−NH3 –CH4 O−CO]+ [M+H−NH3 –CH4 O−CH4 O]+ [M+H−NH3 –CH4 O−CH3 −CO]• +

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

Flavonoids

Standard

Table 1 (Continued) Type

Standard Launobine (6)

selection [M+H]+

Precursor ions 312.12296

Molercular formula C18 H18 O4 N

Error (ppm) −0.239

MSn MS2 [312.12] MS3 [295.10]

MS4 [263.07]

Reflexan A (26)

[M+H]+

439.24750

C27 H35 O5

−0.900

MS2 [439.25]

MS3 [379.23]

MS4 [237.09]

Pyranone II

Katsumadain (27)

[M+H]+

351.19556

C23 H27 O3

0.253

MS2 [351.20]

MS3 [227.07]

MS4 [185.06]

Elem.Comp.

Error (ppm)

Pathways

205.06479 295.09656 280.07303 265.08588 263.07037 235.07530 233.05960 205.06470 397.23730 379.22684 303.12286 263.16415 243.10153 323.16418 297.14835 283.13266 269.11713 263.16388 255.10126 251.10641 243.10132 237.09087 139.03871 129.06970 117.06973 219.08017 209.09584 191.08525 169.10092 129.06970 295.13263 269.11697 255.10130 241.08571 227.07001 215.07005 185.05940 131.04890 203.06999 185.05942 183.04372 157.06448 131.04889 117.06966 157.06456 129.06964 117.06966

C15 H9 O C18 H15 O4 C17 H12 O4 C17 H13 O3 C17 H11 O3 C16 H11 O2 C16 H9 O2 C15 H9 O C25 H33 O4 C25 H31 O3 C17 H19 O5 C16 H23 O3 C15 H15 O3 C21 H23 O3 C19 H21 O3 C18 H19 O3 C17 H17 O3 C16 H23 O3 C16 H15 O3 C17 H15 O2 C15 H15 O3 C16 H13 O2 C7 H7 O3 C10 H9 C9 H9 C16 H11 O C15 H13 O C15 H11 C13 H13 C10 H9 C19 H19 O3 C17 H17 O3 C16 H15 O3 C15 H13 O3 C14 H11 O3 C13 H11 O3 C12 H9 O2 C9 H7 O C12 H11 O3 C12 H9 O2 C12 H7 O2 C11 H9 O C9 H7 O C9 H9 C11 H9 O C10 H9 C9 H9

−0.007 0.253 0.070 −0.154 0.377 −0.239 0.017 −0.446 −0.091 0.181 0.527 −0.080 −0.168 0.028 −0.576 −0.745 −0.338 −1.106 −1.219 −0.981 −1.032 −0.574 −1.874 −1.370 −1.255 −1.239 −1.203 −1.449 −1.520 −1.370 −0.816 −0.932 −1.062 −0.874 −1.148 −1.026 −1.654 −1.842 −1.382 −1.546 −1.836 −1.983 −1.918 −1.852 −1.474 −1.835 −1.852

[M+H−NH3 –CH4 O−CO−CH4 O]+ [M+H−NH3 ]+ [M+H−NH3 −CH3 ]• + [M+H−NH3 −CH2 O]+ [M+H−NH3 −CH4 O] + [M+H−NH3 –CH4 O−CO] + [M+H−NH3 −CH4 O−CH2 O] + [M+H−NH3 −CH4 O−CH2 O−CO] + [M+H−C2 H2 O] + [M+H−CH3 COOH] + [M+H−C10 H16 ] + [M+H−C11 H12 O2 ] + [M+H−CH3 COOH−C10 H16 ] + [M+H−CH3 COOH−C4 H8 ] + [M+H−CH3 COOH−C6 H10 ] + [M+H−CH3 COOH−C7 H12 ] + [M+H−CH3 COOH−C8 H14 ] + [M+H−CH3 COOH−C9 H8 ] + [M+H−CH3 COOH−C9 H16 ] + [M+H−CH3 COOH−C8 H14 −H2 O] + [M+H−CH3 COOH−C10 H16 ] + [M+H−CH3 COOH−C9 H16 −H2 O] + [M+H−CH3 COOH−C10 H16 −C8 H8 ] + [M+H−CH3 COOH−C15 H22 O3 ] + [M+H−CH3 COOH−C16 H22 O3 ] + [M+H−CH3 COOH−C9 H16 −H2 O−H2 O] + [M+H−CH3 COOH−C9 H16 −H2 O−CO] + [M+H−CH3 COOH−C9 H16 −H2 O−CO−H2 O] + [M+H−CH3 COOH−C9 H16 −H2 O−C3 O2 ] + [M+H−CH3 COOH−C9 H16 −H2 O−C6 H4 O2 ] + [M+H−C4 H8 ]+ [M+H−C6 H10 ]+ [M+H−C7 H12 ]+ [M+H−C8 H14 ]+ [M+H−C9 H16 ]+ [M+H−C10 H16 ]+ [M+H−C9 H16 −C2 H2 O]+ [M+H−C12 H18 O−C2 H2 O]+ [M+H−C9 H16 −C2 ]+ [M+H−C9 H16 −C2 H2 O]+ [M+H−C9 H16 −C2 H4 O]+ [M+H−C9 H16 −C2 −H2 O−CO]+ [M+H−C9 H16 −C5 H4 O2 ]+ [M+H−C9 H16 –C5 H2 O3 ]+ [M+H−C9 H16 –C2 H2 O−CO]+ [M+H−C9 H16 –C2 H2 O–2CO]+ [M+H−C9 H16 –C2 H2 O−C3 O2 ]+

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

Pyranone I

Fragment ions

39

40

Table 2 MSn data and fragmentation pathways for the compounds identified from L. reflexa Hemsl. Compound No.

tR (min)

selection

Measured mass

Molercular formula

Error (ppm)

MSn m/z

Type

1*

3.07

[M+H]+

314.13870

C18 H20 O4 N

−0.049

Alkaloids

2**

3.21

[M+H]+

286.14359

C17 H20 O3 N

−0.629

3*

3.31

[M+H]+

314.13855

C18 H20 O4 N

−0.428

4** 5**

3.44 3.76

[M+H]+ [M+H]+

300.15945 328.15408

C18 H22 O3 N C19 H22 O4 N

0.100 −0.776

6*

4.57

[M+H]+

312.12296

C18 H18 O4 N

−0.239

7**

5.62

[M+H]+

300.15933

C18 H22 O3 N

−0.300

8**

5.67

[M+H]+

286.14368

C17 H20 O3 N

−0.315

9**

5.81

+

[M+H]

328.15408

C19 H22 O4 N

−0.776

10*

7.63

[M±NH4 ]+

554.22247

C26 H36 O12 N

−1.321

11***

10.83

[M+H]+

535.21545

C27 H35 O11

−3.621

14.07

+

213.09085

C14 H13 O2

−0.733

+

MS2 [314.14]: 297.11188; MS3 [297.11]: 282.08847, 265.08572; MS4 [265.09]: 250.06227, 237.09079, 233.05986, 222.06696, 205.06465; MS2 [286.14]: 269.11716; MS3 [269.12]: 237.09082, 209.09593, 175.07512, 145.06459, 137.05954, 107.04898. MS2 [314.14]: 297.11230; MS3 [297.11]: 282.08887, 265.08609; MS4 [265.09]: 250.06256, 237.09106, 233.05986, 222.067, 205.06479. MS2 [300.16]: 269.11716; MS3 [269.12]: 237.09082, 209.09602, 175.07526, 145.06461. MS2 [328.15]: 297.11209; MS3 [297.11]: 282.08853, 265.08585; MS4 [265.09]: 250.06235, 237.09082, 233.05957, 205.06459. MS2 [312.12]: 295.09656; MS3 [295.10]: 280.07303, 265.08588, 263.07037; MS4 [263.07]: 235.07530, 233.05960, 205.06470. MS2 [300.16]: 269.11707; MS3 [269.12]: 237.09076, 209.09573, 175.07507, 145.06454, 143.04903, 137.05945. MS2 [286.14]: 269.11707; MS3 [269.12]: 237.09077, 209.09612, 175.07507, 145.06458, 143.04892, 137.05948; MS4 [237.09]: 209.09576. MS2 [328.15]: 297.11185; MS3 [297.11]: 282.08832, 265.08548; MS4 [265.09]: 250.06235, 237.09053, 233.05939, 205.06441. MS2 [554.22]: 537.19598, 375.14331, 213.09073; MS3 [213.09]: 135.04384, 107.04894, 195.08017, 167.08522, 91.05414; MS4 [135.04]: 107.04895. MS2 [535.22]: 389.15869, 373.16400, 371.14844, 353.13803, 227.10614; MS3 [227.11]: 209.09573, 195.08034, 181.10062, 149.05931, 121.06454. MS2 [213.09]: 195.08047, 167.08546, 135.04402, 107.0490, 91.05415; MS3 [135.04]: 107.04890. MS2 [257.08]: 239.06999, 215.07008, 179.03363, 173.05945, 153.01797, 145.06458, 131.04895, 103.05405. MS2 [283.10]:265.08554, 205.04927, 192.04147, 153.01802, 131.08533, 91.05406. MS2 [281.08]: 263.07043, 225.09143, 153.01794, 129.06967, 115.05392. MS2 [227.11]: 209.09595, 195.08034, 181.10110, 167.08534, 149.05959, 121.06473, 91.05412; MS3 [149.06]: 121.06464. MS2 [439.25]: 397.23706, 379.22638, 263.16370; MS3 [379.23]: 323.16370, 297.14844, 269.11673, 255.10121, 243.10118, 237.09068. MS2 [365.21]: 347.20004; MS3 [347.20]: 291.13696, 265.12210, 225.09061, 213.09073; MS4 [291.14]: 213.09100; MS4 [213.09]: 195.07999, 167.08504, 135.04375, 107.04887. MS2 [271.10]: 253.08598, 229.08598, 193.04945, 173.05959, 167.03380, 131.04909, 103.05415. MS2 [397.24]: 379.22647, 361.21573, 263.16391, 243.10130; MS3 [379.23]: 283.13272, 269.11685, 255.10126, 243.10127, 117.06976. MS2 [397.24]: 379.22638, 263.16382, 255.10085, 243.10123; MS3 [379.23]: 255.10095, 243.10030, 117.06957. MS2 [349.22]: 293.15317, 239.10626, 225.09062, 213.09071, 137.13220; MS3 [213.09]: 195.08025, 167.08536, 135.04396, 107.04904. MS2 [393.21]: 289.14313, 283.09604, 269.08041, 257.08051, 179.03362,131.04881; MS3 [269.08]: 251.07011, 227.06979, 165.01787. MS2 [351.20]: 295.13278, 269.11685, 241.08549, 227.06993, 215.06976, 185.05930; MS3 [227.07]: 185.05943; MS4 [185.06]: 117.06995. MS2 [439.25]: 397.23691, 379.22627, 263.16412, 243.10136; MS3 [379.23]: 323.16330, 283.13184, 269.11691, 255.10123, 251.10664, 243.10139, 237.09068, 209.09586, 117.06958; MS4 [237.09]: 209.09605.

[M+H]

13*

15.94

[M+H]

257.08072

C15 H13 O4

−0.449

14*** 15*** 16*

16.83 17.61 17.70

[M+H]+ [M+H]+ [M+H]+

283.09644 281.08081 227.10681

C17 H15 O4 C17 H13 O4 C15 H15 O2

−0.161 −0.090 0.677

17***

18.26

[M+H]+

439.24759

C27 H35 O5

−0.707

18*

19.80

[M+H]+

365.21048

C24 H29 O3

−1.756

19*

20.00

[M+H]+

271.09665

C16 H15 O4

0.607

20***

20.33

[M+H]+

397.23657

C25 H33 O4

−1.928

21***

20.53

+

[M+H]

397.23709

C25 H33 O4

−0.619

22***

20.98

[M+H]+

349.21570

C24 H29 O2

−1.451

23***

21.51

[M+H]+

393.20514

C25 H29 O4

−2.278

24***

21.91

+

[M+H]

351.19540

C23 H27 O3

−0.203

25***

22.42

[M+H]+

439.24765

C27 H35 O5

−0.559

Alkaloids Alkaloids Alkaloids Alkaloids Alkaloids Alkaloids Alkaloids Stilbenes Stilbenes Stilbenes Flavonoids Flavonoids Flavonoids Stilbenes Pyranone I Stilbenes Flavonoids Pyranone I Pyranone I Stilbenes Flavonoids Pyranone II Pyranone I

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

12*

Alkaloids

Table 2 (Continued) Compound No.

tR (min)

selection

Measured mass

Molercular formula

Error (ppm)

MSn m/z

Type

26*

22.67

[M+H]+

439.24750

C27 H35 O5

−0.900

Pyranone I

27*

22.87

[M+H]+

351.19556

C23 H27 O3

0.253

28***

23.27

[M+H]+

439.24768

C27 H35 O5

−0.502

29***

23.47

[M+H]+

351.19516

C23 H27 O3

−0.886

30***

23.68

+

[M+H]

439.24771

C27 H35 O5

−0.422

31***

23.93

[M+H]+

351.19547

C23 H27 O3

−0.003

32***

24.14

[M+H]+

303.12268

C17 H19 O5

−0.067

33*

24.35

[M+H]+

349.21622

C24 H29 O2

0.038

34***

24.40

[M+H]+

351.19537

C23 H27 O3

−0.288

35***

24.97

[M+H]+

349.21622

C24 H29 O2

0.038

36***

25.27

[M+H]+

351.19531

C23 H27 O3

−0.459

37***

26.02

[M+H]+

439.24762

C27 H35 O5

−0.627

38***

26.32

+

[M+H]

393.20575

C25 H29 O4

−0.727

39***

26.45

[M+H]+

439.24762

C27 H35 O5

−0.627

40***

26.80

[M+H]+

407.22098

C26 H31 O

−1.734

41***

26.94

+

[M+H]

393.20599

C25 H29 O4

−0.117

42***

27.08

[M+H]+

351.19540

C23 H27 O3

−0.203

MS2 [439.25]: 397.23730, 379.22684, 263.16415, 243.10153; MS3 [379.23]: 323.16418, 297.14835, 283.13266, 269.11713, 263.16388, 255.10126, 251.10641, 243.10132, 237.09087, 139.03871, 129.06970, 117.06973; MS4 [237.09]: 219.08017, 209.09584, 191.08525, 169.10092, 129.06970. MS2 [351.20]: 295.13263, 269.11697, 255.10130, 241.08571, 227.07001, 215.07005, 185.05940, 131.04890; MS3 [227.07]: 203.06999, 185.05942, 183.04372, 157.06448, 131.04889; MS4 [185.06]: 157.06456, 129.06964, 117.06966. MS2 [439.25]: 397.23685, 379.22641, 263.16388, 243.10129; MS3 [379.23]: 323.16373, 297.14801, 283.13245, 269.11679, 263.16345, 255.10124, 251.10643, 243.10124, 237.09067, 129.06964, 117.06959; MS4 [237.09]: 219.08023, 209.09583, 191.08524, 169.10085, 129.06966. MS2 [351.20]: 295.13248, 269.11697, 255.10056, 241.08562, 227.06996, 215.06998; MS3 [227.07]: 185.05940, 131.04881; MS4 [185.06]: 117.06957. MS2 [439.25]: 397.23679, 379.22638, 263.16385, 243.10130; MS3 [379.23]: 323.16388, 297.14819, 283.13260, 269.11691, 263.16360, 255.10123, 251.10628, 243.10124, 237.09070, 139.03854, 129.06961, 117.06965; MS4 [237.09]: 219.08009, 209.09589, 191.08521, 169.10085, 129.06966. MS2 [351.20]: 295.13263, 269.11661, 255.10132, 241.08566, 227.06999, 215.06995, 185.05942, 131.04875; MS3 [227.07]: 203.06993, 185.05937, 183.04359, 157.06439, 131.04886, 117.06963; MS4 [185.06]: 157.06473, 129.06975, 117.06966. MS2 [303.12]: 261.11185, 243.10135, 127.03878; MS3 [243.10]: 201.09068, 197.09581, 183.08017, 179.08533, 169.10100, 165.06960, 155.08528, 139.03876, 131.08533, 117.06976. MS2 [349.22]: 293.15332, 239.10658, 225.09076, 213.09073, 137.13222; MS3 [213.09]: 195.07990, 167.08525, 135.04367, 107.04881. MS2 [351.20]: 295.13257, 269.11688, 255.10127, 241.08559,227.06998, 215.06998, 185.05936, 131.04890; MS3 [227.07]: 203.06990, 185.05936, 183.04370, 157.06450, 131.04884, 117.06965; MS4 [185.06]: 157.06432, 129.06958, 117.06960. MS2 [349.22]: 293.15329, 239.10634, 225.09068, 213.09070, 137.13214; MS3 [213.09]: 195.08022, 167.08537, 135.04390, 107.04902. MS2 [351.20]: 241.08589, 227.06993, 215.06995; MS3 [215.07]: 197.05940, 187.07500, 169.06433, 131.04886; MS4 [187.08]: 169.06439, 143.04871, 129.06964, 117.06966. MS2 [439.25]: 397.23691, 379.22635, 263.16388, 243.10124; MS3 [379.23]: 323.16333, 297.14761, 269.11688, 263.16376, 255.10132, 243.10141, 237.09085, 117.06958. MS2 [393.21]: 289.14297, 283.09604, 269.08032, 257.08060, 179.03343, 131.04889; MS3 [269.08]: 251.06912, 227.07016, 165.01785. MS2 [439.25]: 379.22634, 303.12241, 261.11188, 243.10120; MS3 [303.12]: 261.11191; MS4 [261.11]: 243.10114, 127.03880. MS2 [407.22]: 351.15857, 325.14279, 303.15897, 297.11163, 283.09598, 271.09567, 179.03351, 131.04875; MS3 [283.10]: 241.08528, 179.03339. MS2 [393.21]: 289.14346, 283.09619, 269.08054, 257.08047, 179.03331, 131.04907; MS3 [269.08]: 251.07018, 227.07001, 165.01785. MS2 [351.20]: 295.13269, 269.11713, 255.10106, 241.08562, 227.07002, 215.07007, 185.05939, 131.04922; MS3 [227.07]: 203.06982, 185.05946, 131.04883, 117.06973; MS4 [185.06]: 129.06993, 117.06980.

Pyranone II

Pyranone II Pyranone I

Pyranone II

Pyranone I Stilbenes Pyranone II

Stilbenes Pyranone II

Pyranone I Flavonoids

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

Pyranone I

Pyranone I Flavonoids Flavonoids Pyranone II

*Confirmed by comparing with the reference compounds; **compounds have been reported in literatures; ***potential new compounds.

41

42

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

Fig. 2. Total ion chromatogram (TIC) of L. reflexa Hemsl. in positive ion mode using UHPLC-LTQ-Orbitrap-MS.

a

H+

H+

H+

-C6H6

m/z 135.04 (C8 H7 O2)

m/z 135.04 (C8 H7 O2)

m/z 213.09 (C14 H13 O2) -H2O

-CO -CO

b H+

-C7H6O2

w-27109-1-30v33 #1187 RT: 14.03 AV: 1 NL: 2.50E6 T: FTMS + c ESI d Full ms2 [email protected] [50.00-225.00] 135.04395 100 90

m/z 195.08 (C14 H11 O)

80

m/z 107.05 (C7 H7 O)

70 60 50

-CO

40

H+

30

m/z 91.05 (C7 H7)

20

107.04903

10 79.05414 91.05405

0 60

m/z 167.09 (C13 H11)

80

100

121.02826 120

141.06966 140 m/z

195.08025 213.09085

167.08537 160

180

200

220

Fig. 3. The fragmentation pathway of pinosylvin recorded using a collision energy of 30 V (a) and its CID MS/MS spectrum (b).

introduced in the positive ion modes of the ESI source. Because of their remarkably higher abundance in the positive ion mode, the [M+H]+ or [M±NH4 ]+ ions were selected as the precursor ions for CID fragmentation to produce the MS/MS spectra. Then, the prominent MS/MS product ions were selected for further MSn analysis (n = 3–4). The fragmentation pathways and MSn mass spectra of pinosylvin (compound 12) are shown in Fig. 3. The [M+H]+ ion at m/z 213.0909 (C14 H13 O2 ) for pinosylvin produced a prominent product ion at m/z 135.0440 (C8 H7 O2 ) through the typical loss of the C6 H6 group. The minor ion at m/z 195.0805 (C14 H11 O) was attributed to the loss of H2 O, which was also a main product ion for m/z 213.0909. Notably, the loss of CO was also apparent in the MS3 spectra at m/z 195.0805 (m/z 195.0805 → 167.0855) and at m/z 135.0440 (m/z 135.0440 → 107.0491). It is proposed that the hydroxyl on C-3 was rearranged first in the A-ring. Compounds 10,

33 and 18 are pinosylvin derivatives and showed similar fragmentation behaviors as compound 12. The typical loss of 78 Da (C6 H6 ), 28 Da (CO), 18 Da (H2 O) and 28 Da (CO) was commonly observed, and all produced the ions at m/z 213.0907 (C14 H13 O2 ), 195.0803 (C14 H11 O), 167.0855 (C13 H11 ), 135.1166 (C14 H13 O2 ) and 107.0490 (C7 H7 O). Due to the existence of a methoxy group at C-3, the fragment ions of 3-methoxy-5-hydroxy-trans-stilbene (compound 16) were m/z 227.1068 (C15 H15 O2 ), m/z 209.0960, m/z 181.1011, m/z 149.0596, and m/z 121.0646. The characteristic ions of compound 16 were 14 Da larger than others in this group. Meanwhile, compound 11 gave rise to a [M+H]+ ion at m/z 535.2155 (C27 H35 O11 ), and the MS/MS spectrum showed ions at m/z 373.1640 (C21 H25 O6 ) and 227.1061 (C15 H15 O2 ) by consecutive loss of a terminal glucose and a deoxyglucose. The ions at m/z 209.0957 (C15 H13 O), m/z 181.1006 (C14 H13 ), m/z 149.0593 (C9 H9 O2 ), and

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

m/z 121.0645 (C8 H9 O) indicated that compound 11 was a derivative of compound 16. This compound was tentatively identified as 3-O-glucose-deoxyglucose-2-methoxy-trans-stilbene, which was a new compound. Compounds 22 and 35 show the same [M+H]+ ions at m/z 349.2157 (C24 H29 O2 ), and the ions at m/z 213.0907 (C14 H13 O2 ), 195.0803 (C14 H11 O), 167.0855 (C13 H11 ), 135.1166 (C14 H13 O2 ), and 107.0490 (C7 H7 O) indicated that they had similar fragmentation behaviors as reflexanbene I (compound 33). Therefore, these compounds were identified as the isomers of reflexanbene I in the extracted ion chromatogram. 3.2.2. Flavonoids In the present study, 2 standards, pinocembrin (compound 13) and pinostrobin (compound 19), were identified and used to characterize the fragmentation behaviors and explore the fragmentation rules for the flavonoids, as shown in Fig. 4a–c. The CID pathways observed for these standards exhibit some common features. The fragmentations that were most useful for identifying the flavonoids were those involving cleavage of the two C C bonds at positions 1/3, 1/4, and 2/4 of the C ring and the C C bonds at 5, resulting in structurally informative i,j A+ and i,j B+ ions (Fig. 4a). The fragmentation route is designated in the pathways shown in Fig. 4b and c. For example, pinocembrin (compound 13) gave rise to 1.3 A+ ions at m/z 153.0180 (C7 H5 O4 ), 1.4 B+ ions at m/z 131.0490 (C9 H7 O), 2.4 A+ ions at m/z 215.0701 (C H O ) and 5 A+ ions at m/z 179.0336 13 11 3 (C9 H7 O4 ) in the fragmentation pathways. Pinostrobin (compound 19) gave rise to 1.3 A+ ions at m/z 167.0338 (C8 H7 O4 ), 1.4 B+ ions at m/z 131.0491 (C9 H7 O), 2.4 A+ ions at m/z 229.0860 (C14 H13 O3 ) and 5 A+ ions at m/z 193.0495 (C H O ) in the fragmentation pathways. 10 9 4 The subsequent loss of H2 O, CO, RDA and C-ring fragmentation were the most possible fragmentation pathways for flavonoids. The [M+H]+ ion at m/z 283.0964 (C17 H15 O4 ) for compound 14 gave rise to 1.3 A+ ions at m/z 153.0180 (C7 H5 O4 ), 1.4 B+ ions at m/z 131.0490 (C9 H7 O) and 5 A+ ions at m/z 205.04927 (C11 H9 O4 ), indicating that it is a flavonoid. Compound 15 gave rise to 1.3 A+ ions at m/z 153.0179 (C7 H5 O4 ) and 1.3 B+ ions at m/z 129.0697 (C10 H9 ), indicating that it is also a flavonoid. Compound 23 has [M+H]+ ions at 393.2051 (C25 H29 O4 ), and the MS/MS spectrum showed an ion at m/z 257.0805 (C15 H13 O4 ) through the loss of a terminal p-mentheneyl (C10 H16 ), an ion at m/z 269.0804 (C16 H13 O4 ) through the loss of C9 H16 , 1.3 A+ ions at m/z 289.14307 (C17 H21 O4 ), 1.4 B+ ions at m/z 131.0488 (C9 H7 O), 2.4 A+ ions at m/z 227.0700 ([M+H−C9 H16 −C2 H2 O]+ ) from the fragment ions at m/z 269.0804 ([M+H−C9 H16 ]+ ) and 5 A+ ions at m/z 179.0335 (C9 H7 O4 ) in the fragmentation pathways. Therefore, compound 23 was tentatively identified as p-mentheneyl-pinocembrin in the extracted ion chromatogram. Compounds 38 and 41 have the same [M+H]+ ions at 393.2058 (C25 H29 O4 ), and their MSn spectra showed similar fragmentation behaviors as p-mentheneylpinocembrin. Therefore, they were tentatively identified as isomers of p-mentheneyl-pinocembrin. Compound 40 has [M+H]+ ions at 407.2210 (C26 H31 O4 ), and the MS/MS spectrum showed an ion at m/z 271.0957 (C16 H15 O4 ) through the loss of a terminal p-mentheneyl (C10 H16 ), an ion at m/z 283.0960 (C17 H15 O4 ) through the loss of C9 H16 , 1.3 A+ ions at m/z 303.1590 (C18 H23 O4 ), 1.4 B+ ions at m/z 131.0488 (C H O), 2.4 A+ ions at m/z 241.0853 9 7 ([M+H−C9 H16 −C2 H2 O]+ ) from the fragment ions at m/z 283.0960 ([M+H−C9 H16 ]+ ) and 5 A+ ions at m/z 193.04906 (C10 H9 O4 ) in the fragmentation pathways. Therefore, compound 40 was tentatively identified as p-mentheneyl-pinostrobin. 3.2.3. Alkaloids The alkaloids consisted of norbracteoline (compound 1), laetanine (compound 3), and launobine (compound 6). The MSn mass spectrometry data and the fragmentation pathways of laetanine are shown in Fig. 5a and b. The precursor ion at m/z 314.1386

43

(C18 H20 O4 N) produced a [M+H−NH3 ]+ ion at 297.1123 (C18 H17 O4 ) through the loss of NH3 . [M+H−NH3 ]+ could be further fragmented to produce the ion [M+H−NH3 −CH4 O]+ at m/z 265.0861 (C17 H13 O3 ). Then, the fragment ions at m/z 265.09861 could be fragmented to produce the ion at m/z 237.0911 (C16 H13 O2 ) through the loss of CO. Norbracteoline (compound 1), which is the isomer of laetanine, showed the same [M+H]+ ions at 314.1387 (C18 H20 O4 N) and similar fragmentation behaviors as laetanine. The [M+H]+ ion at m/z 312.1230 (C18 H18 O4 N) for launobine (compound 6) produced a [M+H−NH3 ]+ ion at m/z 295.0966 (C18 H15 O4 ) through the loss of NH3 . [M+H−NH3 ]+ could be further fragmented to produce the ion [M+H−NH3 −CH4 O]+ at m/z 263.0703 (C17 H11 O3 ). Then, the fragment ions at m/z 263.0703 could be fragmented to produce the ion at m/z 235.0753 (C16 H11 O2 ) through the loss of CO. Therefore, the successive loss of 17 Da (NH3 ), 32 Da (CH4 O) and 28 Da (CO) were critical data for interpreting the fragmentation pathway of alkaloids. Two isomers (compounds 2 and 8) generated the [M+H]+ ions at 286.14359 (C17 H20 O3 N). Then, successive loss of 17 Da (NH3 ), 32 Da (CH4 O) and 28 Da (CO) from the [M+H]+ ion indicated that these compounds belonged to the group of alkaloids. Therefore, they were tentatively identified as isococlaurine and coclaurine [25,26]. Compounds 4, 5, 7 and 9 featured the characteristic loss of 31 Da (CH5 N, indicating the presence of a methyl group at N2). Then, successive loss of 32 Da (CH4 O) and 28 Da (CO) suggested that these compounds belong to the group of alkaloids. Compounds 4 and 7 shared the same [M+H]+ ions at 300.1595 (C18 H22 O3 N) and displayed similar fragmentation patterns. Thus, compounds 4 and 7 were tentatively identified as N-methylisococlaurine and N-methylcoclaurine [27,28]. Compound 5 and 9 gave rise to the [M+H]+ ions at 328.1541 (C19 H22 O4 N). These two compounds showed ions at m/z 297.1121, 265.0859, and 237.0908 through the consecutive loss of CH5 N, CH4 O and CO. Based on previous reports [29,30], they were tentatively characterized as boldine and isoboldine. 3.2.4. Pyranone I In this study, the UHPLC-LTQ-Orbitrap-MS method was applied for the first time to identify the fragmentation behaviors of reflexan A (compound 26), which was determined to possess a novel skeleton. The novel skeleton that connected the 4-phenyl-2-acetoxy group-n-butyl derivative to a pyran-2-one group [1] was defined as a pyranone I type skeleton. The MSn mass spectrometry data and the fragmentation pathways of reflexan A are shown in Fig. 6a and b. The full scan mass spectrum of reflexan A gave a [M+H]+ ion at m/z 439.2475 (C27 H35 O5 ). Neutral loss of C2 H2 O and C2 H4 O2 generated ions at m/z 397.2473 (C25 H33 O4 ) and 379.2368 (C25 H31 O3 ), respectively. The ion at m/z 243.1015 (C15 H15 O3 ) was generated from the ion at m/z 379.2368 through the loss of a p-mentheneyl group. In the MS3 spectrum, the base peak at m/z 379.2368 (C25 H31 O3 ) was further fragmented to lose a molecule of C9 H16 , H2 O and CO to yield ions at m/z 255.1013 (C16 H15 O3 ), m/z 237.0909 (C16 H13 O2 ) and m/z 209.0958 (C15 H13 O), respectively. These ions were useful for predicting the novel skeletons. Although there is a lack of sufficient reference compounds, some compounds corresponding to the novel skeleton were detected by comparing the fragmentation behavior of reflexan A. Compounds 17, 25, 28, 30, 37 and 39 shared the common ion [M+H]+ at m/z 439.2475 (C27 H35 O5 ). The presence of ions at m/z 397.2473, m/z 379.2368, m/z 255.1013, and m/z 243.1013 indicated that these compounds shared the same skeleton with reflexan A and were isomers of reflexan A. Two isomeric compounds, compounds 20 and 21, both showed [M+H]+ precursor ions at m/z 397.2366 (C25 H33 O4 ), and common fragmentation behaviors, as the ions at m/z 379.2265, 255.1013 and 243.1013 in the MS2 spectrum were observed during their ESI-MSn fragmentations, suggesting that

44

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

a

b

1,4B+

1,3A+

H+

H+

-H2O

-C8H8

1,3A+

m/z 153.02 (C7 H5 O4)

m/z 239.07 (C15 H11 O3)

m/z 257.08 (C15 H13 O4) -C6H6

-C6H6O3

-C2H2O

5A+

2,4A+

H+

c 5A+ w-27109-1-30v33 #1341 RT: 15.87 AV: 1 NL: 1.11E6 T: FTMS + c ESI d Full ms2 [email protected] [60.00-270.00] 153.01801 100

1,4B+

m/z 179.03 (C9 H7 O4)

m/z 131.05 (C9 H7 O)

90

2,4A+

80 70

m/z 215.07 (C13 H11 O3)

-CO

131.04895

-C2H2O

60 50

H+

40

-CO

30 173.05945

20 10 0

215.07008 179.03363

103.05402 123.04397 73.89399 91.50214 60

80

100

120

239.06999 257.08072 140

160 m/z

180

200

220

240

260

m/z 145.06 (C10 H9 O)

m/z 173.06 (C11 H9 O2)

m/z 103.05 (C8 H7)

Fig. 4. The fragment behaviors of flavonoids under low-energy CID: nomenclature and diagnostic product ions of the protonated flavonoids (a).The fragmentation pathway of pinocembrin recorded using a collision energy of 30 V (b) and its CID MS/MS spectrum (c).

b a

w-27109-1-30v33 #283 RT: 3.32 AV: 1 NL: 3.05E7 T: FTMS + c ESI d Full ms2 [email protected] [75.00-325.00] 297.11240

100 90 80 70

-NH3

60

-CH3

50 40 30 20 265.08606

10

m/z 314.14 (C18 H20 O4 N)

m/z 297.11 (C18 H17 O4 )

m/z 282.09 (C17 H14 O4 )

-CH4O

88.55848

0

134.90331 158.88185

199.25650

150

200 m/z

100

237.08342

282.08789

250

300

w-27109-1-30v33 #284 RT: 3.33 AV: 1 NL: 1.77E7 T: FTMS + c ESI d Full ms3 [email protected] [email protected] [70.00-310.00] 265.08609

100 90 80

-CH3

70

-CH4O

60 50 40 30 297.11237

20

m/z 250.06 (C16 H10 O3 )

m/z 265.09 (C17 H13 O3 )

m/z 233.06 (C16 H9 O2 )

10 0

78.85453 80

117.72975 145.66096 167.13058 100

120

140

160

250.06236 205.06483 233.05986

180

200

220

240

282.08887

260

280

300

m/z w-27109-1-30v33 #285 RT: 3.34 AV: 1 NL: 8.83E6 T: FTMS + c ESI d Full ms4 [email protected] [email protected] [email protected] [60.00-280.00]

-CO

265.08603

100

-CO

90 80 237.09106 70

-CH4O

60 50 40 30 20

m/z 222.07 (C15 H10 O2 )

m/z 237.09 (C16 H13 O2 )

m/z 205.06 (C15 H9 O )

10 0

71.45552 88.55884 60

80

100

130.96156 148.64204 120

140

177.06978

160

180

205.06479 222.06755 200

m/z

Fig. 5. The fragmentation pathway of laetanine recorded using a collision energy of 30 V (a) and its CID MSn spectrum (b).

220

250.06256 240

260

280

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

45

a -C2H4O2

m/z 379.23 (C25 H31 O3)

m/z 439.25 (C27 H35 O5)

-C9H16 -C10H16 -C2H2O

H+

m/z 255.10 (C16 H15 O3) -H2O

m/z 243.10 (C15 H15 O3)

m/z 397.24 (C25 H33 O4)

-C8H8 m/z 237.09 (C16 H13 O2)

H+

-CO

-C3O2

m/z 139.04 (C7 H7 O3) m/z 169.10 (C13 H13)

m/z 209. 10 (C15 H13 O)

b w-27109-1-30v33 #1888 RT: 22.70 AV: 1 NL: 6.15E5 T: FTMS + c ESI d Full ms3 [email protected] [email protected] [90.00-390.00] 237.09071 100

w-27109-1-30v33 #1887 RT: 22.69 AV: 1 NL: 4.32E6 T: FTMS + c ESI d Full ms2 [email protected] [110.00-450.00] 379.22641

90

90

80

80 Relative Abundance

Relative Abundance

100

70 60 50 40 30 397.23682

20 225.09088 263.16394

10 0

139.03862 150

205.12207 200

250

300

350

60 50 40 269.11688

30

323.16391 209.09581

20

400

169.10085 117.06963 191.08517 141.06961

10

422.55060 442.13141

275.16367 319.30569 361.21591

255.10126

70

283.13251

379.22644 337.17935

0 100

450

150

200

m/z

250 m/z

300

350

w-27109-1-30v33 #1889 RT: 22.71 AV: 1 NL: 8.22E4 T: FTMS + c ESI d Full ms4 [email protected] [email protected] [email protected] [55.00-250.00] 209.09578 100 90

Relative Abundance

80 70 60

237.09067

169.10078 191.08510

50 40 30 129.06963

20 10

109.02812 71.86691 88.57700

219.08018

181.10068

156.84529 141.06960

0 60

80

100

120

140

160

180

200

220

240

m/z

Fig. 6. The fragmentation pathway of reflexan A recorded using a collision energy of 30 V (a) and its CID MSn spectrum (b).

they represented the reference compound reflexan A lacking C2 H2 O elements. In addition, compound 32 followed similar fragmentation pathways as compound 39. Compound 32 showed [M+H]+ precursor ions at m/z 303.1227 (C17 H19 O5 ) and displayed common fragmentations, such as the ions at m/z 255.1013 and 243.1013 in

the MS2 spectrum, suggesting that compound 39 lacked the C10 H16 elements. 3.2.5. Pyranone II Pyranone II was the skeleton that connected the styryl group to a pyran-2-one group. Katsumadain (compound 27) belongs

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X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

a

H+

H+

H+

m/z 269.12 (C17 H17 O3)

m/z 255.10 (C16 H15 O3)

m/z 295.13 (C19 H19 O3)

-C4H8

-C6H10

H+

-C7H12

H+

H+ -C8H14

-C10H16

m/z 241.09 (C15 H13 O3)

m/z 351.20 (C23 H27 O3) m/z 215.07 (C13 H11 O3) -C14H20O2

-C9H16 -C2H2O

-C5H4O2 m/z 131.05 (C9 H7 O)

m/z 185.06 (C12 H9 O2)

m/z 227.07 (C14 H11 O3)

b W-27109-1-30v33 #1899 RT: 22.84 AV: 1 NL: 1.96E6 T: FTMS + c ESI d Full ms2 [email protected] [85.00-365.00] 227.07001 100

W-27109-1-30v33 #1900 RT: 22.85 AV: 1 NL: 9.94E5 T: FTMS + c ESI d Full ms3 [email protected] [email protected] [50.00-240.00]

90

60 50 40 30 241.08571

20

0

227.06999

80

70

Relative Abundance

Relative Abundance

80

10

185.05942

100

90

100

150

60 50 40 30 20

215.07005 185.05940

92.85968 131.04890

70

255.10130

200

295.13263 309.14810

250

0

350

300

104.67487 88.57706

10

351.18765

55.08095 60

m/z

80

100

131.04889

171.08031

203.06999

157.06448 120

140 m/z

160

209.05971 180

200

220

240

W-27109-1-30v33 #1901 RT: 22.86 AV: 1 NL: 6.46E5 T: FTMS + c ESI d Full ms4 [email protected] [email protected] [email protected] [50.00-200.00] 185.05943

100 90

Relative Abundance

80 70 60 50 40 117.06966

30 20 10 0

54.69072 60

79.08993 80

157.06456

129.06964

105.03335 100

120 m/z

140

160

181.05888 180

199.29793 200

Fig. 7. The fragmentation pathway of katsumadain recorded using a collision energy of 30 V (a) and its CID MSn spectrum (b).

to the pyranone II group and was first isolated from the plants of Lauraceae. In the present study, it was first introduced in the positive ion modes of the ESI source. The MSn mass spectrometry data and the fragmentation pathways of katsumadain are shown in Fig. 7a and b. The full scan mass spectrum of katsumadain gave a [M+H]+ ion at m/z 351.1956, from which the molecular formula was proposed as C23 H27 O3 . Fragment ions at m/z 295.1326 ([M+H−C4 H8 ]+ ), m/z 255.1013 ([M+H−C7 H12 ]+ ) and m/z 241.0857 ([M+H−C8 H14 ]+ ) were thought to be generated from m/z 351.1956 by a series of cleavage and

hydrogen rearrangements occurring in the p-mentheneyl group. A series of ions at m/z 227.0700 ([M+H−C9 H16 ]+ ), m/z 215.0700 ([M+H−C10 H16 ]+ ), m/z 185.0594 ([M+H−C9 H16 −C2 H2 O]+ ) and m/z 131.0489 ([M+H−C9 H16 −C5 H4 O2 ]+ ) corresponded to the skeleton. Compounds 24, 29, 31, 34, 36, and 42 generated the same [M+H]+ ions at 351.1954 (C23 H27 O3 ) in positive mode and four dominant ions at m/z 295.1328 ([M+H−C4 H8 ]+ ), 241.0855 ([M+H−C8 H14 ]+ ), 227.0699 ([M+H−C9 H16 ]+ ), 215.0700 ([M+H−C10 H16 ]+ ), 185.0594 ([M+H−C9 H16 −C2 H2 O]+ ) and 131.0489 ([M+H−C9 H16 −C5 H4 O2 ]+ )

X. Sun et al. / Journal of Pharmaceutical and Biomedical Analysis 126 (2016) 34–47

in the MS2 spectrum. Therefore, they were tentatively identified as isomers of katsumadain. 4. Conclusions An efficient and sensitive method employing ultra-high performance liquid chromatography coupled with linear trap quadrupole orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS) was developed for the qualitative analysis of chemical constituents of LR in the positive ion mode. By combining a wide range of information, including the exact mass from the LTQ-Orbitrap-MS, the fragmentation patterns, the retention time on UHPLC, and comparing the results with reference substances and the literature, a total of 42 compounds, including 8 stilbenes, 8 flavonoids, 9 alkaloids, 10 pyranone I compounds and 7 pyranone II compounds, 24 of which are potentially new compounds, were detected from LR. This is the first report on the chemical constituents in LR using UHPLC-LTQOrbitrap-MS. The results not only provide abundant information for the identification and better understanding of the chemical constituents of LR but also provide beneficial information for further pharmacokinetic studies, metabolic studies and quality control of this traditional Chinese herb. The study also suggested that UHPLCLTQ-Orbitrap-MS would be a powerful and reliable analytical tool for the characterization of chemical profiles in complex chemical systems, such as herbal medicines and TCM preparations, although further investigations are still needed to confirm the absolute configurations of the isomers. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2012ZX09103201-024) and Innovation Scientists and Technicians Troop Construction Projects of Henan Province (Grant No. 114200510014). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2016.04.023. References [1] S.Q. Chen, L.L. Wang, W.Q. Zhang, Y.L. Wei, Secondary metabolites from the root of Lindera reflexa Hemsl, Fitoterapia 105 (2015) 222–227. [2] Y.M. Luo, L.Q. Huang, P. Wang, Q. Li, Study on the volatile oil in aerial part and underground part of Lindera reflexa Hemsl, Chin. Pharm. J. 39 (2004) 307–309. [3] L.D. Zhou, J. Guo, J.G. Yu, Flavonoids of Beijing propolis, China. J. Chin. Mater. Med. 24 (1999) 162–164. [4] J.Z. Zhang, Q.C. Fang, Application of high speed counter-current chromatography to the separation of stilbene derivatives from the roots of Lindera reflexa, Planta Med. 60 (1994) 190–191. [5] J.W. Lei, Y.L. Guo, Y. Bai, S.Q. Chen, Establish the quantitative model of 3,5-didroxystilbene in Lindera reflexa Hemsl by near-infrared spectroscopy, Chin. J. Exp. Tradit. Med. Form. 17 (2011) 75–77. [6] Koon-Sin Ngo, Geoffrey D. Brown, Stilbenes, monoterpenes, diarylheptanoids, labdanes and chalcones from Alpinia katsumadai, Phytochemistry 47 (1998) 1117–1123.

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