Chemical Fingerprinting of Medicinal Plants ‘‘Gui-jiu’’ by LC-ESI Multiple-Stage MS
2008, 68, 781–789
Yan Zhou1,2, Shun-Yuan Jiang3, Li-Sheng Ding2, Sau-Wan Cheng1, Hong-Xi Xu4, Paul Pui-Hay But1,&, Pang-Chui Shaw1,& 1
2 3 4
Institute of Chinese Medicine and Departments of Biology and Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, People’s Republic of China; E-Mail:
[email protected];
[email protected] Chengdu Institute of Biology, Chinese Academy of Sciences, 610041 Chengdu, People’s Republic of China Sichuan Institute of Chinese Materia Medica, 610041 Chengdu, People’s Republic of China Hong Kong Jockey Club Institute of Chinese Medicine, Shatin, Hong Kong, People’s Republic of China
Received: 4 April 2008 / Revised: 25 June 2008 / Accepted: 15 July 2008 Online publication: 30 August 2008
Introduction
Abstract The rhizomes or roots of Dysosma versipellis (Hance) M. Cheng, Dysosma pleiantha (Hance) Woodson, and Sinopodophyllum emodi (Wall. Ex Royle) Ying under the same name ‘‘Guijiu’’, were widely used for medicinal purposes. Valid quality control of Gui-jiu is desirable due to the fact that they are highly toxic. In the present paper, an accurate fingerprint method was developed to identify the three species. Liquid chromatography coupled with photodiode array detection and electrospray ionization-multiple stage mass spectrometry were developed to identify the fingerprint components. According to the characteristic fragmentation behavior of known lignans and flavonoids isolated from D. versipellis, a total of 15 constituents in the crude extracts were structurally characterized on the basis of retention time, UV and tandem mass spectrometric analysis. Eight batches of D. versipellis, three batches of D. pleiantha, and two batches of S. emodi were collected from different locations and these representative samples reflected the similar chemical constituent properties. The extracts were separated by a C18 reversed phase LC column, with a gradient solvent system. The proposed method provides a scientific and technical platform to the herbal industry for quality control and safety assurance of herbal preparations that contain this class of poisonous podophyllotoxin-type lignans.
Keywords Liquid chromatography-mass spectrometry Electrospray ionization-multiple stage mass spectrometry Chemical fingerprinting Dysosma versipellis, Dysosma pleiantha and Sinopodophyllum emodi
Original DOI: 10.1365/s10337-008-0786-0 0009-5893/08/11
The rhizomes of Dysosma versipellis (Hance) M. Cheng, Dysosma pleiantha (Hance) Woodson, and Sinopodophyllum emodi (Wall. Ex Royle) Ying are referred to as ‘‘Giu-jiu’’ in China. It is commonly used for the treatment of rheumatoid arthritis with numbness of the limbs, and pyogenic infection of skin tissue [1–3]. Chemically, Gui-jiu contains various lignans, flavonoids and steroids. The lignans are found to be the main components [4–15]. These lignans have attained considerable interest because of their antitumor [16], antimitotic [17], antiviral [18] and insecticidial activities [19]. For instance, etoposide and teniposide are both semi-synthetic glycoside derivatives of podophyllotoxin, with good clinical perspectives against several types of cancer [20, 21]. Since these podophyllotoxin related lignans are highly toxic compounds, the use of Guijiu as a herbal medicine is potentially dangerous and needs to be carefully controlled. Therefore, chemical identification of Gui-jiu by using this class of aryltetralin-type lignans as marker compounds is essential.
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OH OH HO
HO
O
HO
H
O
OH
OH
HO OMe
OH
OH
O
HO H
O HO
O
H
H HOHO
HO HO HO O
HOHO
H
(1)
HO OCH3
OH O
O HO H
O
O OH
O O O
OCH3
HO
O
HO HO
OCH3
O
O
(3)
Chemicals and Reagents
O
O
O
Experimental
H
(2)
O
O
O OH H
OH H
HO OMe
O
HO
HO H
O OH H
H HOHO
MeO
OMe
MeO
H
OMe OMe
OH (4)
(6)
(5) OH
OH O O
HO
O
O
OH
O
O O
OH
O
O OH OH
MeO
O
MeO
OMe
OMe OMe
OH
(9)
(8)
(7)
OH O
O O
O
O
HO
O
ESI–MSn was summarized, which was applied to elucidate other related lignanglycosides in the extracts by on-line LC– ESI–MSn.
OH
O
O
O
LC grade acetonitrile (Tedia, USA) was used for the LC analysis. Water prepared with a Millipore Milli-Q SP purification system (Millipore, France) was used during sample preparation procedures and LC analyses. Dysosmarol (1) [30], 40 -demethyl podophyllotoxin (7), quercetin (8), podophyllotoxin (9), 40 -demethyldeoxy podophyllotoxin (10), kaemferol (11), dehydropodophyllotoxin (12), diphyllin (13) podophyllotoxone (14), and acacetin (15) were chosen as standards (Fig. 1), which were isolated from D. versipellis in our laboratory and were identified based on spectral (NMR and MS) analysis. Their retention times (tR) were 11.8, 26.3, 28.3, 34.6, 35.1, 35.7, 45.1, 46.1, 49.9, and 54.7 min., respectively under the LC conditions as described below.
OH OH
MeO
O
MeO
OMe OH
OMe OMe
(11)
(10)
Collection of Samples
(12)
OH
O
MeO
O O
MeO
O
O
HO
O O
OH O
OMe
O
MeO
O (13)
O
OMe OMe
(15)
(14)
Fig. 1. Structures of the compounds assigned in the extracts of Dysosma versipellis
Previous studies on D. versipellis, D. pleiantha, and S. emodi with thinlayer chromatography [5], gas chromatography-mass spectrometry [22], scanning electron microscopy [23], column chromatography [4–6], DNA techniques [24], and LC [25–28] have been documented. However, LC–MS provides a more accurate means of authentication, as the individual components in the
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fingerprint can be elucidated. Previously, an LC–API–MS method was reported for the identification and characterization of Podophyllum emodi [29]. Our report provides the first establishment of LC–UV–ESI–MS fingerprints for D. versipelli, D. pleiantha, and S. emodi, which are listed as ‘‘Gui-jiu’’. The diagnostic fragmentation of the class of aryltetralin-type lignans by negative
A total of eight samples of D. versipellis, three samples of D. pleiantha, and two samples of S. emodi were collected from different places in China (Table 1). These samples were identified based on morphological and microscopic characteristics. Voucher samples were deposited at the Museum of the Institute of Chinese Medicine, the Chinese University of Hong Kong.
Sample Preparation A small amount of chopped roots (0.5 g) was extracted under supersonic conditions with methanol (10 mL) for an hour. The extracting process was repeated three times. The extraction solutions were combined, filtered, and evaporated under vacuum and diluted to volume with methanol in a 5 mL volu-
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metric flask. The extracted solution was filtered through a 0.22 lm PTFE syringe filter, and an aliquot of each filtrate (10 lL) was injected into the LC instrument for analysis.
Data Analysis of Chromatogram The cosine values of vectorial angle of the entire chromatographic patterns among samples were calculated and the simulative mean chromatogram was calculated using the Computer Aided Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (CASES), a chemometrics computer software developed by the Institute of Pharmacy Engineering (Zhejiang University, Hangzhou, China) and endorsed by the National Pharmacopoeia Committee of the People’s Republic of China for similarity studies of chromatographic fingerprints of Chinese herbs. This software was employed to synchronize the chromatographic peaks, to calculate the cosine values of vectorial angel among different chromatograms, as well as to compute the mean chromatogram as a representative standard fingerprint/ chromatogram for a group of chromatograms. The cosine values of the two chromatograms approaching 1 means they are highly similar. The standard LC fingerprint is set up with the median of all chromatograms [31–33]. The similarities of the entire chromatographic profiles were analyzed among the tested samples. The relative retention time (RRT) and relative peak area (RPA) of each characteristic peak to reference peak were calculated in the chromatograms.
Computational Method All calculations were carried out using the Gaussian 03 program package [34]. The B3LYP hybrid density functional and 6-31G (d) basis set were used. For all optimized structures, vibrational spectra were calculated to ensure that no
Original
Table 1. Collected crude rhizomes of D. versipellis, D. pleiantha and S. emodi Samples
Species
Location of collection
Voucher specimen
DV-1 DV-2 DV-3 DV-4 DV-5 DV-6 DV-7 DV-8 DP-1 DP-2 DP-3 SE-1 SE-2
D. versipellis D. versipellis D. versipellis D. versipellis D. versipellis D. versipellis D. versipellis D. versipellis D. pleiantha D. pleiantha D. pleiantha S. emodi S. emodi
Hongqing Shizhu Yunnan Dongshan Yunnan Luliang Zhejiang Panan Zhejiang Dapanshan Jiangxi Wuzhou Anhui Haozhou Zhejiang Ningbo Guangxi Guilin Jiangxi Fuliang Sichuan Mianyang Anhui Huangshan Sichuan Aba
2006-2989 2006-2990 2006-2991 2006-2992 2006-2985 2006-2986 2006-2987 2006-2988 2006-2993 2006-2994 2006-2996 2006-2997 2006-2998
Table 2. The LC–MSn data in negative mode of the methanol extract of D. versipellis, D. pleiantha, and S. emodi No.
tR (min)
[M–H](m/z)
MS/MS (m/z)
MS3 (m/z)
Identification
1
11.8
375
297
Dysosmarol
2
14.9
463
345(100), 327, 297 301
271, 179, 151
3
16.0
447
4
18.2
561
5 6 7 8
22.5 22.9 26.4 28.3
577 575 399 301
Quercetin 3-O-glucopyranoside Kaempferol 3-O-glucopyranoside 40 -Demethylpodophyllotoxin glucoside Podorhizol-b-D-glucoside Podophyllotoxin glucoside 40 -Demethylpodophyllotoxin Quercetin
9 10
34.3 35.1
413 383
11 12 13
35.7 45.1 45.8
285 409 379
14
49.9
411
15
54.7
283
327, 285(100), 257 399 415 413(100), 395 384 257, 179(100), 151 383 368(100), 351 257, 241, 151 394, 364, 350 364(100), 334, 319, 305 367(100), 352, 337 268
151 384 400, 383, 371 383, 327 369, 300 151 368, 327 353 379 319
Podophyllotoxin 40 -Demethyldeoxy podophyllotoxin Kaemferol Dehydropodophyllotoxin Diphyllin
352
Podophyllotoxone
240
Acacetin
imaginary frequencies for energy minimum were obtained.
Equipment and Chromatographic Conditions An LC system consisting of a vacuum degasser, quaternary pump, autosampler and PAD detector (Thermo Separation Products Inc., Riviera Beach FL, USA) was used for acquiring chromatograms and UV spectra. Chromatographic separation was achieved using a C18 column (250 9 4.6 mm, 5 lm; Diamonsil,
USA), and a gradient solvent system comprised of 0.1% formic acid-water (solvent A) and acetonitrile (solvent B); the gradient profile was: 0–15 min, linear 15–30% B; 15–45 min, linear 30–50% of B; 45–60 min, linear 50–70% B; 60– 65 min, linear 70–15% of B, with a flow rate of 0.8 mL min-1. UV detection was at 270 nm. The LC effluent was split so that approximately 400 lL min-1 effluent entered the ESI source. ThermoQuest Finnigan LCQDECA system equipped with an atmospheric ionization source (ThermoQuest LC–MS Division, San Jose, CA, USA) was used
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and six replicates of sample extraction. Precision of retention times and peak areas of the ten standard compounds for replicated injection were in the range of 0.17–1.3% and 1.4–3.0% of RSD (n = 6), respectively. The peak area RSD of the ten standard compounds in sample replicates were about 2.5– 4.3% (n = 6). The limit of detection (LOD) (S/N = 3) of the ten standard compounds ranged from 1.61 to 38.5 ng. The results indicated that the conditions for the fingerprint analysis were satisfactory. After 65 min, no peaks were found; chromatograms within 65 min were studied.
Results and Discussion Selection of Chromatographic Conditions Fig. 2. Tandem mass spectra of 40 -demethyl podophyllotoxin (7) OH
OH
O O O O MeO
OMe
-CO
O O
-CH3
O O MeO
O 399
OH
O -CH3
-CO2
O O
O
O
O 384
341
O
O O 369
RDA
O
-CO2-H2O
325 307
-CO2-H2O-CO 279
O
O 285 MeO
O O 300
Fig. 3. Fragmentation pathways of 40 -demethyl podophyllotoxin (7)
for mass spectrometric measurement. The ESI–MSn spectra were acquired in both, the positive and negative ion modes. The mass spectrometry detector (MSD) parameters were as follows: capillary temperature 250 °C; spray voltage 4.5 kV; capillary voltage, -15 V in (-) ESI, 24 V in (+) ESI, lens voltage, 18 V in (-) ESI, -16 V in (+) ESI; full scan (m/z 100–1000) using 500 ms for collection time; sheath gas flow 60 arbitrary units of nitrogen; auxiliary gas flow 14 arbitrary units of nitrogen; the optimized relative collision energies of 35–45%. The
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precursor ion isolation window was set at 5 Th to maximize signal/noise in the fragment ion spectra; the scan range was m/z 50–1,000. All data acquired were processed by Finnigan Xcalibur core data system Rev. 1.2 (ThermoQuest Corporation, San Jose, CA, USA).
Method Validation Method reproducibility and repeatability were evaluated by analyzing six injections of the same extracted solution
In this study, most compounds of interest are podophyllotoxin related lignans (Fig. 1). Podophyllotoxin (9) was often used as marker compound owing to its bioactivity and presence in relatively higher content [26–28]. Although quercetin (8) and kaemferol (11) were demonstrated previously as major components in D. versipellis, they were not chosen as marker compounds for assessing the quality of D. versipellis before. In this work, both podophyllotoxin-type lignans (1, 7, 9, 10, 12–14) and flavonoids (8, 11 and 15), which belong to different classes of compounds, were first chosen as characteristic compounds for detection. Selection of detection wavelength was one of the key factors contributing to a reliable and reproducible LC fingerprint of D. versipellis. The wavelength of 207 and 285 nm were used for detection of podophyllotoxin [22–25], while 250–285 and 350–370 nm were often used for detection of flavonoid compounds including quercetin and kaemferol [28]. Our LC-PAD chromatograms showed that D. versipellis could be differentiated from D. pleiantha and S. emodi with the marker compounds diphyllin and dysosmarol. Also, the maximum UV absorption for diphyllin and dysosmarol was at 270 nm, at which most of the other compounds in the
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chromatograms also possessed strong absorbance. Hence, detection at 270 nm was adopted.
383.1
100 50 188.9
0
Original
368.1
20
Relative Abundance
413.2
382.4
305.1 329.2
36
Fragmentation Behavior of Pure Lignans The authentic samples of lignan, namely dysosmarol (1), 40 -demethyl podophyllotoxin (7), podophyllotoxin (9), 40 -demethyldeoxy podophyllotoxin (10), dehydropodophyllotoxin (12), diphyllin (13) podophyllotoxone (14), which were isolated from D. versipellis, were studied by means of (–)ESI–MSn. Their structures are depicted in Fig. 1 and the tandem mass data are shown in Table 2. All lignans could be analyzed in the negative ion mode, and were detected as deprotonated molecules. Most of the lignans (compounds 7, 10, 12–13) gave the primary fragmentation product ion [M–H–15]- presumably by the loss of a methyl radical. While compounds 1 and 9 gave [M–H–30]-, compound 14 gave [M–H–44]-as their primary product ion (MS2), which can be explained by the loss of CH2O or CO2, respectively. But they also showed the losses of a methyl radical upon further fragmentation (MS3-5). The result indicated that the loss of a methyl radical was the most commonly observed fragmentation behavior (MS2–MS5) of all podophyllotoxin related lignans containing methoxyl groups. 40 -Demethyl podophyllotoxin (7) as an example of aryltetralin-type lignan, gave the base peak [M–H–15]- at m/z 384 in MS2 when using the deprotonated molecule [M–H]- at m/z 399 as the precursor ion. In MS3 the precursor ion at m/z 384 gave the product ions at m/z 369 and 300, which were formed by the loss of a methyl radical, and by retro Diels-Alder (RDA) cleavage, respectively. Further fragments were detected in the MS4 spectrum from the m/z 369 ion, which were formed by losses of CO, CO2, CO2 + H2O, CO2 + H2O + CO. The tandem mass spectra are shown in Fig. 2 and the fragmentation mechanism is proposed in Fig 3. In contrast, podophyllotoxin (9) showed a base peak of [M–H–30]- at
235.3 261.0
327.0 194.1 208.4
0
364.9
325.2
382.6
353.1
45 20
339.1 353.9
200.3
0
325.0 338.1
8 5
321.1
293.2 153.1
353.2
280.8
215.2 239.2
0 310.1
2
320.1 239.1 309.1 253.2 268.1 201.8 223.4
1 0
337.3
310.1
2 1 240.3 224.9
0 100
150
200
309.4 281.2 293.2
250
325.3 325.9
300
350
400
450
500
m/z Fig. 4. Tandem mass spectra of podophyllotoxin (9) OH -H O
O
-CH3
O O
O
-CO
O O MeO
OMe OMe 413
-OCH2
O MeO
-CH3
-CH3
368
353 -CH3
OMe OMe
310
325
-56 Da
383
-H2O
320
-CO
310
338
327
Fig. 5. Fragmentation pathways of podophyllotoxin (9)
m/z 383 in MS2 spectrum, which was derived by the loss of the CH2O unit. It should be pointed out that the elimination of the neutral fragment CH2O might correspond to three possible pathways. The first is attributable to form C-1–O–C2 bond; the second is via cleavage the C-7– C-8 bond, and the C-8–C-9 bond to form a five member B ring; the third is formed through elimination of the CH2O unit from the methoxy unit. We have calculated the relative energies among these three structures, and found structure 2 is
more stable than structures 1 and 3. The energy of structure 1 is 220.0 kJ mol-1 higher than structure 2 and structure 3 is 10.9 kJ mol-1 higher than structure 2. The precursor ion at m/z 383 showed successive loss of methyl radical in MS3-5. The proposed fragmentation mechanism is shown in Fig. 5. Thus, the number of losses of methyl radical could be used for these lignans to identify the number of methoxyl groups. The tandem mass spectra are shown in Fig. 4.
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Fig. 6. LC chromatograms of the crude extracts from 8 samples of D. versipellis (DV-1–8)
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Identification of Lignans in Fingerprint Chromatograms
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On-line LC–MSn was utilized to determine the constituents in the crude extracts of D. versipellis. The fragmentation rules of the podophyllotoxin-type lignans can be used to characterize similar structures from the crude extracts. The LC profiles of D. versipellis included peaks 1 and 15 and are shown in Fig. 6. Ten of the peaks were unambiguously identified as dysosmarol (1), 40 -demethyl podophyllotoxin (7), quercetin (8), podophyllotoxin (9), 40 -demethyldeoxy podophyllotoxin (10), kaemferol (11), dehydropodophyllotoxin (12), diphyllin (13) podophyllotoxone (14), and acacetin (15) by comparison the retention times, UV and ESI–MSn spectra with authentic standards (Table 2). For those peaks with no available standards for reference, the identities were assigned by comparing the retention time and molecular weight of chemicals in literature, and by interpretation of MS–MS spectra, as discussed below. For peak 4, a deprotonated molecular ion [M–H]- at m/z 561 suggested its molecular weight is 562; the strong negative fragment ion at m/z 399 [M–H– 162]- in MS2 spectrum proved that the glucosyl unit was easily lost, and the diagnostic fragment ion at m/z 399 then followed fragmentation pathways of 40 demethylpodophyllotoxin (7) up to MS5. For example, in MS3 spectra, fragmentation of the ion of m/z 399 yielded the product ions at m/z 384 by losing a methyl radical. In MS4 spectra the product ions at m/z 369 and 300 were formed from the precursor ion at m/z 384 by the loss of a methyl radical, and by RDA cleavage. Referring to the literature, it could be tentatively elucidated as 40 -demethylpodophyllotoxin glucoside [13–15]. For peak 5, the deprotonated molecular ions [M–H]- at m/z 577 was found in the ESI–MS spectra; the characteristic fragment ions at m/z 415 [M–H–162]- in tandem mass spectra suggested that one glucosyl unit was easily lost and the fragmention of m/z 415 then produced fragment ions at m/z 400 [M–H–162–CH3]-, 383 [M–H–
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162–CH3OH]-, 371 [M–H–162–CO2]-. By comparison with literature, it could be tentatively elucidated as podorhizol b-D-glucoside [35]. For peak 6, the deprotonated molecular ions [M–H]- at m/z 575 indicated its molecular weight is 576; the characteristic fragment ion at m/z 413 [M–H–162]- also displayed the loss of one glucosyl moiety, and the fragment ion of m/z 413 exhibited fragmentation pathways of podophyllotoxin up to MS5. For example, in MS3 spectra fragmentation of the ion of m/z 413 yielded the product ions at m/z 383, derived by the loss of the CH2O unit; m/z 383 in turn lost a methyl radical to form the product ions at m/z 368, 353, and 338 in MS4-6, respectively. Therefore, it could be tentatively elucidated as podophyllotoxin glucoside [13–15].
Identification of Flavonoids in Fingerprint Chromatograms Quercetin, kaemferol and their glycosides were commonly found in D. versipellis extracts and its related species. The spectra of flavonoid derivatives in D. versipellis species were characterized. Both flavonoids and their glycosides show similar UV spectra, which usually comprise the strong absorption bands with maxima around 250–265 and 350– 360 nm. Since the shoulder absorption of these flavonoids is characteristically different from the podophyllotoxin-type lignans, the use of a wavelength above 300 nm is preferred in the analysis of the flavonoids. Compounds (8) and (11) are contained as major components, while 15 were also found in all the tested samples, and their identification was achieved by a direct comparison with standards. Other peaks, without authentic samples, were identified by a combination of UV, MS data and reference of literature. Peak 2, with UV absorption at 256, 265 and 357 nm, represented the longest wavelength of all flavonol derivatives discussed here. Its MS spectrum showed a [M–H]- ion at m/z 463, the MS2 spectrum has a dominant peak at m/z 301 corresponding to [M–H–162]-,
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Time (min) Fig. 7. Base peaks trace of LC–MS analysis of the crude extracts from three samples of D. pleiantha (DP-1–3), 2 samples of S. emodi (SE-1–2)
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Table 3. The relative retention time (RRT) and relative peak area (RPA) of characteristic peaks Peak no.
D. versipellis RRT
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.693 ± 0.874 ± 0.882 ± 0.897 ± 0.920 ± 0.941 ± 0.946 ± 0.964 ± 1 1.007 ± 1.013 ± 1.251 ± 1.341 ± 2.041 ± 2.588 ±
D. pleiantha RPA
0.0045 0.0028 0.0016 0.0027 0.0061 0.0072 0.0082 0.0078 0.015 0.011 0.045 0. 021 0.067 0.055
0.255 ± 0.632 ± 0.724 ± 0.162 ± 1.056 ± 0.539 ± 0.974 ± 0.458 ± 1 0.056 ± 1.076 ± 0.596 ± 0.768 ± 0.077 ± 0.178 ±
which was formed by the elimination of a glucosyl unit. The ion at m/z 301 follows identical fragmentation pathways as quercetin (8) up to MS4 [36]. Peak 3 showed UV absorption at 269, 303 and 353 nm. Its MS spectrum showed a [M–H]- ion at m/z 447, and cleavage of the glucosidic bond formed the ion at m/z 285 in MS–MS spectra. The fragment ion of m/z 285 follows fragmentation pathways similar to kaemferol (11) up to MS4 [36]. Therefore, peaks 2 and 3 are proposed to be quercetin-3-O-glucopyranoside and kaemferol-3-O-glucopyranoside, which have previously been isolated from S. emodi [37].
LC Fingerprints of D. versipellis, D. pleiantha, and S. emodi. To standardize the fingerprint, eight samples of the rhizomes of D. versipellis were analyzed. These samples were collected from different locations. Peaks that existed in all eight samples were assigned as ‘‘characteristic peaks’’ for rhizomes of D. versipellis. There are 15 characteristic peaks (from peak 1 to peak 15) in the fingerprints (Fig. 7). CASES were used to evaluate these chromatograms. Among these peaks, peaks 9 and 11 were chosen as markers for peak matching and peak 9 was chosen to
788
RRT 0.13 0.26 0.38 0.16 0.44 0.24 0.28 0.39 0.25 0.41 0.39 0.28 0.043 0.14
0.559 ± 0.763 ± 0.907 ± 0.914 ± 0.946 ± 0.959 ± 0.973 ± 0.989 ± 1 1.009 ± 1.016 ± 1.067 ± 1.330 ± 1.749 ± 2.103 ±
S. emodi RPA
0.0007 0.0036 0.0026 0.0048 0.0039 0.0078 0.0024 0.0008 0.0013 0.0006 0.0027 0.0032 0.0072 0.013
0.269 ± 0.624 ± 0.639 ± 0.558 ± 0.125 ± 0.891 ± 0.865 ± 0.574 ± 1 0.150 ± 0.637 ± 0.396 ± 0.162 ± 0.052 ± 0.169 ±
RRT 0.26 0.047 0.024 0.086 0.93 1.05 0.39 0.337 0.32 0.35 0.41 0.0504 0.32 0.35
calculate RRT and RPA. RRT and RPA of characteristic peaks in the eight samples are shown in Table 3. The data indicated that the amount of these compounds was similar in D. versipellis but different in other related species. Therefore, the simulative mean chromatograms of the eight test samples of D. versipellis can be used for differentiating D. versipellis from D. pleiantha and S. emodi. Three samples of D. pleiantha were compared to those of D. versipellis. The correlation coefficient of D. pleiantha chromatogram to their simulative mean chromatogram was 0.97 ± 0.0384 (mean ± SD, n = 4), and the correlation coefficient of the simulative mean chromatogram of D. versipellis to that of D. pleiantha was 0.85. There was no unique characteristic peak in their chromatograms for distinguishing D. versipellis from D. pleiantha. Nevertheless, for D. versipellis, the peak intensity of compound (4) was low while that of compounds (5) and (13) was high as compared with D. pleiantha. Therefore, D. versipellis might be differentiated from D. pleiantha through these peaks. Two samples of S. emodi were analyzed, and the correlation coefficient of each chromatogram to their simulative mean chromatogram was 0.93 ± 0.0315 (mean ± SD, n = 2). However, the chromatogram of S. emodi is different from that of D. versipellis (Figs. 6 and 7).
– 0.362 ± 0.441 ± 0.515 ± 0.658 ± 0.719 ± 0.759 ± 0.818 ± 1 1.016 ± 1.032 ± 1.174 ± 1.327 ± 1.393 ± 1.588 ±
RPA
0.0034 0.0023 0.0016 0.012 0.015 0.0004 0.0010 0.0007 0.0020 0.0016 0.0050 0.0022 0.0026
– 0.155 0.367 0.283 0.089 0.441 0.231 0.102 1 0.049 0.219 0.472 0.257 0.045 0.066
± ± ± ± ± ± ±
0.025 0.13 0.11 0.12 0.14 0.057 0.41
± ± ± ± ± ±
0.23 0.46 0.38 0.033 0.10 0.093
The correlation coefficient of the simulative mean chromatogram of S. emodi to that of D. versipellis was 0.79, and to D. pleiantha was 0.77 (Table 3). For the fifteen compounds, compound (1) does not exist or present in a trace amount in the S. emodi chromatograms and the amount of other compounds are in general less than those in D. versipellis and D. pleiantha.
Conclusions In this study, fragmentation behavior of some podophyllotoxin-type lignans was summarized using negative ion ESI–MSn spectra, and the corresponding fragmentation rules were proposed, which can be beneficial to obtain complementary structural information for identification of related lignans. By combining ESI–MSn and LC–ESI–MSn spectra, podophyllotoxin related glucoside, and flavonoid glycosides in the crude extract can be rapidly and effectively characterized. Moreover, the LC–PAD–MSn method was used for fingerprinting analysis of the rhizomes of ‘‘Gui-jiu’’, a toxic herbal material in Chinese medicine. The chromatograms of eight batches of D. versipellis samples with 15 compounds identified were found to be consistent as evaluated by CASES. The simulative mean chromatogram can therefore be
Chromatographia 2008, 68, November (No. 9/10)
Original
employed as the standard fingerprint of D. versipellis. The developed LC fingerprints and the different peak areas of compounds (1), (4), (5), and (13) can be used to differentiate D. versipellis from D. pleiantha and S. emodi.
Acknowledgments Partial support was received from the Hong Kong Jockey Club Charities Trust Fund.
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