Journal of Chromatographic Science 2015;53:886– 897 doi:10.1093/chromsci/bmu137 Advance Access publication October 29, 2014
Article
Rapid Identification and Simultaneous Quantification of Multiple Constituents in Nao-Shuan-Tong Capsule by Ultra-Fast Liquid Chromatography/Diode-Array Detector/Quadrupole Time-of-Flight Tandem Mass Spectrometry Panlin Li1, Weiwei Su1, Chengshi Xie2, Xuan Zeng1, Wei Peng1 and Menghua Liu1,3* 1
Guangzhou Quality R&D Center of Traditional Chinese Medicine, Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, PR China, 2Guangdong Zhongsheng Pharmaceutical Company Limited, Dongguan 523325, PR China, and 3School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China *Author to whom correspondence should be addressed. Email:
[email protected] Received 23 May 2014; revised 8 September 2014
A rapid and high-sensitive ultra-fast liquid chromatography coupled with a diode-array detector and a quadrupole/time-of-flight mass spectrometry (MS) method was established and validated for the chemical profiling of Nao-shuan-tong capsule (NSTC) and simultaneous quantification of five major constituents. A total of 59 components including monoterpene glycosides, flavonoids, sesquiterpenoids, ketosteroids, thiophenes, organic acids and alkaloids were identified or tentatively characterized in NSTC based on the accurate mass and tandem MS behavior. Five major bioactive constituents were chosen as the chemical indexes of holistic quality evaluation and quantified simultaneously. All calibration curves showed good linear regression (r2 > 0.9991) in the range 25.2– 510, 145 – 2,900, 1.84 – 36.8, 2.61 – 52.2 and 3.25 – 26.2 mg/mL for gastrodin, paeoniflorin, typhaneoside, b-ecdysterone and isorhamnetin3-O-neohesperidoside, respectively. It also showed good precision, stability and accuracy for quantification of these five compounds. The limit of detections and limit of quantitations for the analytes ranged from 0.14 to 1.09 mg/mL and from 0.47 to 3.63 mg/mL, respectively. The validated quantification method was applied to analyze 10 batches of commercial NSTC. These results will provide a basis for quality control of the production process and the further pharmacological study in vivo of NSTC.
Introduction Nao-shuan-tong capsule (NSTC) is approved as an ischemic stroke drug by the state Food and Drug Administration of China (state medical license no. Z20040093) with remarkable therapeutic effects (1). It is a kind of Chinese herbal medicine compound preparation (CHMCP) according to the meridian theory of traditional Chinese medicine, comprising Typhae Pollen (TP), Paeoniae Radix Rubra (PR), Curcumae Radix (CR), Gastrodiae Rhizoma (GR) and Rhapontici Radix (RR). It is well known that the complex interaction between compounds may produce synergistic effects and reduce possible side effects. However, the CHMCP that consists of several crude drugs often contains so many poorly characterized chemical constituents that it takes a great challenge to standardize the production process and understand their action mechanisms. In the previous research, the major constituents in these single herbs have been studied partly, for instance, TP mainly contained flavonoids such as naringenin and typhaneoside (2). Different kinds of monoterpene glycosides such as paeoniflorin and benzoylpeaoniflorin were found in PR (3). Sesquiterpenoids were one of the characteristic constituents responsible for the therapeutic effect in CR
(4). Gastrodin in GR and ecdysteroids in RR were commonly used as quality control markers, respectively (5, 6). However, to the best of our knowledge, the profile of chemical constituents in NSTC has not been investigated so far. Recently, a liquid chromatography (LC) system coupled with diode-array detector (DAD) – time-of-flight (TOF) – mass spectrometry (MS) has become an important tool to analyze compounds in the traditional Chinese medicine, a complex mixture containing hundreds of different chemical constituents (7 – 9). In the present study, an ultra-fast liquid chromatography method on a reversed-phase column was developed for high-speed separation of multiple constituents in NSTC. The fragmentation mechanisms of authentic standards including monoterpene glycosides, flavonoids, sesquiterpenoids, ketosteroids, organic acids and alkaloids were first characterized by TOF–MS/MS in both positive and negative modes. Then, the identification and elucidation of the complex constituents of the NSTC were carried out by comparing the retention times, DAD spectra and TOF-MS/MS data of each analyzed compound with those of the corresponding authentic standard and/or the literatures. A total of 59 constituents were unambiguously identified or tentatively characterized in the formula. In addition, simultaneous quantification of five major compounds was developed and 10 batches of commercial NSTC were analyzed, which will provide comprehensive information for quality control and pharmacology research of NSTC.
Experimental Samples, chemicals and reagents Ten batches (nos 100501, 101201, 110302, 110603, 110604, 110701, 110702, 110703, 110704 and 110801) of NSTC and its raw drugs including TP (Typha angustifolia L.), PR (Paeonia lactiflora Pall.), CR (Curcuma wenyujin Y.H.Chen et C. Ling), GR (Gastrodia elata Bl.) and RR (Rhaponticum uniflorum DC.) were provided by Guangdong Huanan Pharmaceutical Group Co., Ltd (Guangdong, China). The plant species were identified by Prof. Wenbo Liao from Sun Yat-Sen University. The voucher specimens were deposited in our laboratory. The authentic standards of paeoniflorin, isorhamnetin-3O-neohesperidoside, typhaneoside, b-ecdysterone, apigenin, isorhamnetin, gallic acid, gastrodin, protocatechuic acid, catechin, vanillic acid, chlorogenic acid, choline chloride, adenine, adenosine, phenylalanine and germacrone were purchased from the National Institute for the Control of Pharmaceutical and
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email:
[email protected]
Biological Products (Beijing, China) with purity .98% detected by high performance liquid chromatography (HPLC). Kaempferol3-O-neohesperidoside, quercetin-3-O-neohesperidoside, quercetin-3-glucoside, calycosin, naringenin, syringic acid, L-pyroglutamic acid and guanosine were purchased from Sigma-Aldrich (St Louis, USA) with purity .95% detected by HPLC. Acetonitrile of HPLC grade was purchased from Fisher Scientific (Pittsburgh, PA, USA). All water used was distilled and further purified by a Milli-Q system (Millipore, Milford, MA, USA). Other reagents used in the experiment were of analytical grade.
Preparation of standard solutions, NSTC and raw drug extractions The mixed solution of 25 standards for identification was prepared in methanol at the concentration of 0.1 mg/mL for each compound. The mixed standard solutions of gastrodin, paeoniflorin, typhaneoside, b-ecdysterone and isorhamnetin3-O-neohesperidoside for quantification were prepared at 0.510, 2.90, 0.0368, 0.0522 and 0.0262 mg/mL, respectively, for their various concentrations in NSTC samples. Then, a series of working solutions of appropriate concentrations was obtained by diluting the mixed standard solution. The content of NSTC (2.5 g) was bath-sonicated twice at 40 kHz for 30 min in 50 mL of methanol, and 5 mL of extract solution was transferred into a 10-mL volumetric flask and diluted up to the mark with methanol. The extracts of T. angustifolia L. (5.6 g), P. lactiflora Pall. (4.0 g), C. wenyujin Y.H.Chen et C. Ling (3.2 g), G. elata Bl. (1.6 g) and R. uniflorum DC. (2.4 g) were separately prepared according to the technological process as the patent report ( patent no.: CN 1491705A). Then, the merged solution was evaporated under reduced pressure at 608C with a rotary evaporator, and the residue was redissolved in 10 mL of methanol. Before analysis, all solutions mentioned above were centrifuged at 15,000 rpm (21,130 g) in an Eppendorf 5424R centrifuge for 10 min. The supernatants were then transferred to an autosampler vial for ultra-fast liquid chromatography/diode-array detector/quadrupole time-of-flight tandem mass spectrometry (UFLC –DAD –Q-TOF –MS/MS) analysis.
UFLC– DAD–Q-TOF –MS/MS system Analysis was performed with a Shimadzu UFLC XR instrument (Shimadzu Corp., Kyoto, Japan) equipped with an in-line degasser, a binary pump, an autosampler, a column oven and a DAD. Chromatographic separation was carried out on a Phenomenex Kinetex C18 column (2.1 100 mm, 2.6 mm, Phenomenex, CA, USA) at 308C. The mobile phase consisted of 0.1% formic acid (v/ v) in both acetonitrile (A) and water (B) using a gradient elution program of 2% A (0 –5 min), linear gradient from 2 to 60% A (5– 20 min), 60 to 100% A (20 – 28 min) and isocratic 100% A for 2 min. The injected volume was 2 mL with the flow rate kept at 0.3 mL/min. The DAD detector scanned from 190 to 400 nm. Detections were performed by a hybrid triple quadrupole time-of-flight mass spectrometer (AB SCIEX Triple TOFTM 5600 plus, AB Sciex Foster City, CA, USA) equipped with electrospray ionization (ESI) source. The TOF –MS worked in a full scan mode and mass range was set at m/z 100 – 1200 in both positive and negative ion modes. The acquisition of MS/MS data was
accomplished by IDA (information-dependent acquisition) mode. The conditions of mass spectrometer were as follows: ion source gas1 55 psi; ion source gas2 55 psi; curtain gas 35 psi; temperature 5508C; ion spray voltage floating 5,500 V; collision energy 40 V; collision energy spread 20 V and declustering potential 80 V. Nitrogen was used as nebulizer and auxiliary gas.
Validation of quantitative analysis The quantification method of multiple constituents in NSTC was established to be specific and suitable for the routine analysis due to its simplicity, sensitivity, accuracy and reproducibility. The bioactive compounds were quantified using DAD chromatograms extracted at 254 nm. The individual compounds were quantified using the calibration curves of external standards. Six different concentrations of the mixed standard working solutions were injected into an UFLC system for the construction of calibration curves. For each target constituent, the limit of detections (LOD) and limit of quantitations (LOQ) were determined at signal-to-noise (S/N) ratios of 3 and 10, respectively. The intra- and inter-day precisions were determined by analyzing six replicates at three concentrations on the same day and three consecutive days in order to test the instrument precision and the method precision. To investigate the stability, the standards and sample extracts were stored in methanol solution at 48C and analyzed every 12 h within 72 h. Accuracy was reported as percent recovery by the assay of known added amount of reference standards in the sample, which were prepared in three different amount levels (80, 100 and 120%) and triplicate experiments at each level. Recoveries were counted by the following formula: Recovery (%) ¼ 100 (amount found 2 original amount)/ amount spiked. Variations were indicated by relative standard deviation (RSD) in all tests.
Results Identification of constituents in NSTC by UFLC–DAD –Q-TOF –MS/MS After the optimization of LC and MS conditions, a simple UFLC– DAD– Q-TOF– MS/MS method was developed to detect constituents in NSTC and its single herb extracts. The DAD chromatogram at 254 nm and base peak chromatogram in positive and negative ion modes are shown in Figure 1. Most of the constituents were well separated under the gradient elution condition with high resolution and good sensitivity. Twenty-five peaks including monoterpene glycosides, flavonoids, sesquiterpenoids, ketosteroids, organic acids and alkaloids were unequivocally identified by comparing their retention times, UV spectra and MS data with those of authentic compounds. When there was no reference standard, the chemical structures were proposed by UV spectra, and molecular mass (mass error ,5 ppm) and fragmentation behaviors obtained from reference standards with the same basic skeleton and available data from literatures. As a result, 34 peaks were tentatively identified. All the chemical structures are presented in Figure 2. The compound profiles of 10 batches of NSTC commercial products were analyzed by UFLC – DAD – Q-TOF – MS/MS, and there was no significant difference in the composition of ingredients as given in Table I. Rapid Identification and Simultaneous Quantification of Multiple Constituents in NSTC 887
Figure 1. DAD chromatogram at 254 nm (A), base peak chromatograms (BPCs) in positive ion mode (B) and negative ion mode (C) of NSTC by UFLC– DAD–Q-TOF– MS/MS. All the peak numbers as in Table I.
Identification of monoterpene glycosides The monoterpene glycosides detected in NSTC are unique compounds originated from PR (3). A total of eight monoterpene glycosides were identified based on their mass spectra and literature reports. All of these compounds showed better responses in negative ion mode. The neutral losses of one formaldehyde molecule (30 Da), one glucosyl moiety (162 Da) or one benzoic acid group (C7H6O2, 122 Da) and ions generated from benzoic acid at m/z 121 ([C7H5O2]2) were usually detected in their MS/MS spectra. Peak 20 displayed the maximum UV absorptions at 229, 277 nm, [M2H]2 ions at m/z 479.1549 (C23H28O11), and yielded fragment ions at m/z 449.1551 and 327.1090 by the successive losses of one formaldehyde molecule (30 Da) and one benzoic acid molecule (122 Da), respectively. Thus, Peak 20 was definitely identified as paeoniflorin by comparing with those of the reference standard. Peak 18 had the same molecular formula with Peak 20 and exhibited [M2H]2 ions at m/z 479.1547 (C23H28O11). It yielded similar fragment ions with Peak 20 at m/z 449.1488 and 327.1097. Another product ions focused on m/z 283.0828 were produced by losing the core moiety (C10H12O4, 196 Da). Thus, Peak 18 was tentatively assigned as albiflorin according to the literature data (7). Peak 12 showed the [M2H]2 ions at m/z 543.1169 (C23H28O11SO2) and was 64 Da (SO2) more than that of Peak 20. Its product ions focused on m/z 421.0809 ([M2H2BA]2) and 259.0259 ([M2H2BA2Glc]2). Therefore, on the basis of 888 Li et al.
occurrence data in P. lactiflora (10), Peak 12 was tentatively identified as paeoniflorin sulfonate. Peak 16 gave the [M2H]2 ions at m/z 495.1500 (C23H28O12) and was 16 Da (O) more than that of Peak 20. It produced fragment ions at m/z 465.1437 ([M2H2HCOH]2) and 333.0985 ([M2H2Glc]2), indicating that Peak 16 may be oxypaeoniflorin based on literature data reported before (7). Peak 27 generated the maximum UV absorptions at 222, 277 nm and [M2H]2 ions at m/z 631.1663, 152 Da (galloyl group, C7H4O4) more than that of Peak 20. It produced fragment ions at m/z 613.1645, 491.1259 and 339.0977 by a series of losses of one H2O molecule, one benzoic acid molecule and one galloyl group, respectively. Refer to the constituents reported in white peony root (10); Peak 27 was tentatively characterized as galloylpaeoniflorin. Peak 39 gave [M2H]2 ions at m/z 599.1764 and produced product ions at m/z 581.1742 ([M2H2H2O]2) and 431.1401 ([M2H2H2O2HCOH2C7H4O2]2). Peak 39 was tentatively identified as benzoyl oxypaeoniflorin, which has been reported in the literature (11). Peak 43 showed [M2H]2 ions at m/z 583.1818 (C30H32O12), and product ions focused on m/z 553.1571 ([M2H2HCOH]2), 461.1408 ([M2H2C7H6O2]2) and 357.1417 ([M2H2HCOH2C10H12O4]2). Thus, Peak 43 was identified as benzoyl paeoniflorin, which has been reported in Radix Paeoniae Rubra (7). Peak 17 exhibited the maximum UV absorptions at 228, 276 nm and [M2H]2 ions at m/z 459.1501 (C20H28O12). Fragment ions at m/z 165.0565, 150.0328 and 122.0394 were
Figure 2. Chemical structures of compounds identified in NSTC. All the peak numbers as in Table I.
obtained by the successive losses of an arabinosylglucose, a methyl group and a carbonic oxide, respectively. Therefore, on the basis of occurrence data in Paeoniaceae (12), Peak 17 was tentatively identified as paeonolide.
Identification of flavonoids Flavonoids have been considered as bioactive principles of many medicinal plants, and showed remarkable antioxidative activities that remain the main topic investigated in recent years (13). In Rapid Identification and Simultaneous Quantification of Multiple Constituents in NSTC 889
890 Li et al.
Table I Identification of the Chemical Constituents of NSTC by UFLC –DAD – Q-TOF– MS/MS No.
TR (min)
[MþH]þ /error (ppm)
1
0.77
104.1069b/20.3
[M2H]2 /error (ppm)
2
1.05
136.0612/23.9
134.0476/3.1
3
1.15
130.0498/20.6
128.0362/7.3
4
1.52
268.1042/0.2
266.0893/20.3
5
1.74
284.0990/0.4
282.0842/20.7
6
1.78
171.0284/22.0
169.0145/2.1
7
2.22
287.1136/3.8
285.0980/0.2
8
2.40
166.0861/20.9
164.0722/3.2
9
3.55
155.0335/22.1
153.0198/3.1
10
5.58
Fragment ions in positive (þ) ion mode 60.0851 [M2C2H4O]þ, 59.0747, 58.0694 [M2C2H6O]þ 119.0363 [MþH2NH3]þ, 92.0264 [MþH2NH3 2HCN]þ, 67.0302, 65.0140 84.0463 [MþH2HCOOH]þ, 56.0537 [MþH2HCOOH2CO]þ 136.0617 [MþH2ribose]þ, 119.0350 [MþH2ribose2NH3]þ 152.0302 [MþH2ribose]þ, 135.0302 [MþH2ribose2NH3]þ, 110.0353 [MþH2ribose2CN2H2]þ 125.0223 [MþH2HCOOH]þ, 109.0272 [MþH2CO2 2H2O]þ, 81.0340 [MþH2CO2 2CO2H2O]þ
120.0808 [MþH2HCOOH]þ, 103.0546 [MþH2HCOOH2NH3]þ, 93.0689 [MþH2HCOOH2NCH]þ, 91.0546 [C7H7]þ, 77.0393 [C6H5]þ
315.0727/1.7 þ
11
6.30
453.1389/20.5
451.1252/1.3
291.0834 [MþH2Glc] , 207.0654 [MþH2Glc22C2H2O]þ, 139.0380, 123.0433
12
6.62
545.1335/2.2
543.1169/21.5
567.1133 [MþNa]þ
13
7.02
291.0864/0.3
289.0725/2.5
147.0438, 139.0388, 123.0439
14
7.28
169.0492/21.9
167.0354/2.7
123.0434 [MþH2HCOOH]þ, 93.0329 [MþH2HCOOH2OCH2]þ
15
7.49
355.1022/20.3
353.0879/0.4
163.0386, 145.0280, 117.0334
16
7.53
17
8.69
461.1651/20.4
459.1501/21.5
18
9.12
481.1703/20.3
479.1547/2.4
495.1500/21.5
167.0964 [MþH2Ara2Glc]þ, 149.0571 [MþH2Ara2Glc2H2O]þ
Fragment ions in negative (2) ion mode
82.0324 [M2H2HCOOH]2, 54.0377 [M2H2HCOOH2CO]2
Formula
Identification
Source
C5H14NO
Cholinea
CR, GR, PR, RR, TP GR, PR, RR, TP
a
C5H5N5
Adenine
C5H7NO3
L-Pyroglutamic
acida
C10H13N5O4
Adenosine
150.0432 [M2H2ribose] , 133.0161 [MþH2ribose-NH3]2, 108.0224 [M2H2ribose2CN2H2]2, 80.0271 [M2H2ribose-CN2H2 2CO]2 125.0253 [M2H2CO2]2, 97.0309 [M2H2CO2 2CO]2, 79.0209 [M2H2CO2 2CO2H2O]2 150.9154 [M2H2C5H10O4]2, 149.9047, 123.0463 [M2H2Glc]2, 105.0363 [M2H2C6H12O6]2 147.0461 [M2H2NH3]2, 103.0547 [M2H2CO2 2NH3]2
C10H13N5O5
Guanosinea
CR, GR, PR, RR, TP CR,PR, RR,TP PR,RR, TP
C7H6O5
Gallic acida
PR
C13H18O7
Gastrodina
GR
C9H11NO2
Phenylalaninea
CR, GR, PR, RR, TP
109.0312 [M2H2CO2]2, 91.0205 [M2H2H2O2CO2]2, 81.0356 [M2H2CO2CO2]2 153.0206 [M2H2Glc]2, 109.0315 [M2H2Glc-CO2]2 289.0753 [M2H2Glc]2, 245.0847 [M2H2Glc2CO2]2, 203.0726 [M2H2Glc2CO2 2C2H2O]2, 151.0410, 125.0255, 109.0312 421.0809 [M2H2BA]2, 259.0259 [M2H2BA2Glc]2, 213.0219 [M2H2BA2Glc2HCOOH]2, 121.0284 [BA2H]2 245.0844 [M2H2CO2]2, 203.0732 [M2H2CO2 2C2H2O]2, 151.0412, 137.0258, 125.0255, 123.0461, 109.0307 152.0126 [M2H2CH3]2, 123.0438 [M2H2CO2]2, 108.0227 [M2H2CH3 2CO2]2 191.0576 [M2H2C6H10O5]2, 173.0468, 135.0463 [M2H2C7H10O5 2CO2]2, 93.0364 465.1437 [M2H2HCOH]2, 333.0985 [M2H2Glc]2, 281.0659 [GlcþOHBA2H2H2O]2, 195.0657 [C10H12O4 2H]2, 137.0247 [OHBA2H]2 293.0891 [AraþGlc2H]2, 165.0565 [M2H2Ara2Glc]2, 150.0328 [M2H2Ara-Glc2CH3]2, 122.0394 [M2H2Ara2Glc2CH3 2CO]2 449.1488 [M2H2HCOH]2, 327.1097 [M2H2HCOH2BA]2, 283.0828 [M2H2C10H12O4]2, 223.0616, 165.0522, 121.0299 [BA2H]2
C7H6O4
Protocatechuic acida
PR
C13H16O9
Protocatechuic acid-3-O-glucoside
PR
C21H24O11
Catechin-7-O-glucoside
PR
C23H28O11SO2
Paeoniflorin sulfonate
PR
C15H14O6
Catechina
PR,TP
C8H8O4
Vanillic acida
PR, TP
C16H18O9
Chlorogenic acida
PR
C23H28O12
Oxypaeoniflorin
PR
C20H28O12
Paeonolide
PR,RR
C23H28O11
Albiflorin
PR
2
a
9.32
199.0601/0.2
197.0457/1.0
20
9.64
481.1700/20.8
479.1549/22.0
21
10.46
757.2179/20.8
755.2085/6.0
22
10.83
789.1110/24.5
787.0996/20.4
23
10.95
611.1610/0.6
609.1494/5.4
465.1064 [MþH2Rha]þ, 303.0498 [M2H2Rha2Glc]þ, 85.0293
24
11.17
741.2221/22.1
739.2141/6.8
25
11.32
771.2341/20.1
769.2260/8.3
26 27
11.63 11.66
465.1028/0.1 633.1814/0
463.0892/2.2 631.1663/20.9
595.1567 449.1046 287.0540 625.1814 479.1165 317.0651 303.0503
28
11.74
595.1652/20.9
593.1539/4.6
Rapid Identification and Simultaneous Quantification of Multiple Constituents in NSTC 891
29
11.87
481.3154/21.3
479.3014/0
30
11.90
625.1757/21.0
623.1645/4.4
31
12.16
32
12.50
435.1285/20.2
433.1131/21.9
33
12.59
625.1757/21.0
623.1625/1.2
34
12.92
479.1179/20.9
35
13.33
171.0293 153.0179 125.0230 107.0137
[MþH2CO]þ, [MþH2HCOOH]þ, [MþH2CO2 2OCH2]þ, [MþH2CO2 2OCH2 2H2O]þ
19
273.0777/3.2
36 37
14.22 14.25
447.0926/0.9 493.1336/20.9
445.0797/4.7 491.1211/3.2
38
14.44
481.3155/21.0
479.3024/2.1
Syringic acida
PR
[M2H2HCOH]2, [M2H2HCOH2BA]2, [M2H2HCOH2BA2C6H10O5]2, [BA2H]2 [M2H22Rha2Glc]2, [M2H22Rha2Glc2CH2O]2
C23H28O11
Paeoniflorina
PR
C33H40O20
Quercetin-3-O-(2G-a-L-rhamnosyl)-rutinoside
TP
617.0902 447.0732 295.0418 169.0140 301.0387 271.0283 179.0000 285.0439 255.0334
[M2H2GA]2, [M2H22GA]2, [M2H22GA2C7H4O4]2, [GA2H]2 [M2H2Rha2Glc]2, [M2H2Rha2Glc2CH2O]2,
C34H28O22
2,3,4,6-tetra-O-galloyl-b-glucose
PR
C27H30O16
Quercetin-3-O-neohesperidosidea
TP
C33H40O19
Kaempferol-3-O-(2G-a-L-rhamnosyl)-rutinoside
TP
C34H42O20
Typhaneosidea
TP
C21H20O12 C30H32O15
Quercetin-3-glucosidea Galloylpaeoniflorin
TP GR,PR
[MþH2Rha]þ, [MþH2Glc2Rha]þ [MþH2H2O]þ, [MþH22H2O]þ, [MþH23H2O]þ, [MþH24H2O]þ, [MþH23H2O2C4H8]þ,
C27H30O15
Kaempferol-3-O-neohesperidosidea
PR,TP
C27H44O7
b-Ecdysterone
[MþH2Rha]þ, [MþH2Glc2Rha]þ
C28H32O16
Isorhamnetin-3-O-neohesperidosidea
CR,TP
C41H32O26
1,2,3,4,6-penta-O-galloyl-b-glucose
PR, RR
C21H22O10
Isosalipurposide
PR
C28H32O16
Isorhamnetin-3-O-rutinoside
CR,TP
C22H22O12
Isorhamnetin-3-O-glucopyranoside
PR,RR,TP
C15H14O5
PR
C21H18O11 C23H24O12
5-Methoxy-2-(3-methoxyphenoxy) benzoic acid Apigenin 7-O-b-glucuronide Quercetin-3,30 -dimethyl ether-40 -glucoside
PR PR
C27H44O7
Ecdysterone isomer
RR
611.16298 [MþH2Rha]þ, 465.1023 [MþH22Rha]þ, 303.0499 [MþH22Rha2Glc]þ, 129.0550
[MþH2Rha]þ, [MþH22Rha]þ, [MþH22Rha2Glc]þ [MþH2Rha]þ, [MþH22Rha]þ, [MþH22Rha2Glc]þ [MþH2Glc]þ, 165.0170
[M2H22Rha2Glc]2, [M2H22Rha2Glc2OCH2]2
613.1645 [M2H2H2O]2, 491.1259 [M2H2H2O2BA]2, 339.0977 [M2H2H2O2BA2C7H4O4]2, 313.0596 [GAþGlc2H2H2O]2, 271.0482, 169.0155[GA2H]2 449.1064 287.0558 463.3029 445.2954 427.2832 409.2748 371.2226 165.1277 479.1187 317.0658
C9H10O5
449.1551 327.1090 165.0557 121.0373 301.0399 271.0278
939.1110/0.2
275.0916/0.7
169.0155 [M2H2CO]2, 125.0274 [M2H2CO2CO2]2
787.1282 769.1003 617.0884 447.0593 259.0248 273.0755 [MþH2Glc]þ, 153.0173, 119.0485 479.1187 [MþH2Rha]þ, 317.0660 [MþH2Glc2Rha]þ 317.0659 [MþH2Glc]þ, 285.0393 [MþH2Glc2CH3OH]þ,153.0178 169.0494 [MþH2C7H6O]þ, 107.0494 þ
271.0603 [MþH2Glucuronide] 331.0823 [MþH2Glc]þ, 313.0652 [MþH2Glc2H2O]þ, 179.0696, 153.0171, 123.0443 463.3067 [MþH2H2O]þ, 445.2959 [MþH22H2O]þ, 427.2846 [MþH23H2O]þ, 409.2940 [MþH24H2O]þ, 311.2022, 299.1649, 281.1529
2
[M2H2C7H4O4] , [M2H2GA]2, [M2H2GA2 C7H4O4]2, [M2H22GA2 C7H4O4]2, [M2H24GA]2, 169.0140
2
167.0355 [M2H2C7H6O] , 123.0454 [M2H2C7H6O2CO2]2 269.0484, 241.0532, 113.0257
a
RR
(continued)
892 Li et al.
Table I Continued [MþH]þ /error (ppm)
[M2H]2 /error (ppm)
No.
TR (min)
39
14.55
40
15.42
285.0761/1.3
41
16.37
237.1848/20.7
42 43
16.80 17.28
273.0757/20.1 585.1960/21.1
271.0617/2.0 583.1818/20.5
44 45
17.39 17.69
271.0602/0.4 265.1436/0.6
269.0459/1.6 263.1295/2.5
46
18.06
317.0658/0.6
315.0517/2.2
47
19.37
231.1378/20.7
48
19.61
231.1378/20.6
49
20.64
359.2219/0.8
50
20.95
217.1586/20.3
51
21.47
219.1742/20.7
52
23.53
235.1693/0.2
53
24.11
219.1744/0.2
54
24.62
237.1849/20.2
55
25.04
249.0038/0.1
Fragment ions in positive (þ) ion mode
599.1764/1.0
283.0618/2.2
357.2085/3.9
233.1545/20.8
246.9895/1.0
270.0518 [MþH2CH3]þ, 253.0494 [MþH2CH3OH]þ, 225.0558 [MþH2CH3OH2CO]þ, 213.0542, 197.0586 [MþH22CO2CH3OH]þ, 137.0226 [1,3A]þ (RDA), 115.0519 219.1824 [MþH2H2O]þ, 191.0289, 161.1388, 135.1128 [MþH2H2O2O2C3H6 2C2H2]þ, 107.0835 153.0180, 147.0430, 91.0536
Fragment ions in negative (2) ion mode
Formula
Identification
Source
581.1742 [M2H2H2O]2, 431.1401 [M2H2H2O2HCOH2C7H4O2]2, 281.0603, 137.0265, 121.0316 [BA2H]2 268.0409 [M2H2CH3]2, 239.0379, 211.0421, 195.0471 [M2H22CO2CH3OH]2, 148.0176, 91.0201
C30H32O13
Benzoyl oxypaeoniflorin
PR
C16H12O5
Calycosina
CR
C15H24O2
Curdione
RR
C15H12O5 C30H32O12
Naringenina Benzoyl paeoniflorin
PR,TP PR
C15H10O5 C15H20O4
Apigenina Zedoarofuran
PR CR,PR
C16H12O7
Isorhamnetina
PR
C15H18O2
Epicurzerenone
CR
C15H18O2
Curzerenone
CR
C22H30O4
Palbinone
PR
C15H20O
Curzerene
CR
C15H22O
b-Turmerone
CR
C15H22O2
13-Hydroxygermacrone
CR
C15H22O
Germacronea
CR,PR
C15H24O2
Curcumol
CR
C12H8O2S2
Arctic acid
RR
553.1571 461.1408 357.1417 121.0303 153.0174, 119.0473, 91.0529 247.1319 [MþH2H2O]þ, 229.1226 [MþH22H2O]þ, 201.1265 [MþH22H2O2CO]þ, 173.1319, 121.1018, 105.0695, 91.0542 302.0440 [MþH2CH3]þ, 274.0488, 165.0161, 153.0199 203.1448 [MþH2C2H4]þ, 185.1334 [MþH2C2H4 2H2O]þ, 155.1016 [MþH2C2H4 2H2O22CH3]þ, 131.0833, 128.0620, 115.0520, 105.0707, 91.0552 203.1402 [MþH2C2H4]þ, 185.1317 [MþH2C2H4 2H2O]þ, 155.0867 [MþH2C2H4 2H2O22CH3]þ, 131.0836, 128.0604, 115.0608, 105.0698, 91.0545 341.2177 [MþH2H2O]þ, 323.2025 [MþH22H2O]þ, 305.1909, 233.1171, 219.1021, 189.1635, 173.0958, 153.0545, 119.0851, 93.0697 161.0927, 131.0850 [MþH2H2O2C5H8]þ, 119.0843, 105.0687 [MþH2H2O2C5H8 2C2H2]þ, 55.0560 177.1268 [MþH2C3H6]þ, 163.1124, 135.1215 [MþH2C3H6 2C2H2O]þ, 119.0818, 107.0864 [MþH2C3H6 2C2H2O2C2H4]þ, 55.0552 217.1583 [MþH2H2O]þ, 199.1508 [MþH22H2O]þ, 189.1653 [MþH2H2O2CO]þ, 157.1001 [MþH22H2O2C3H6]þ, 145.1001, 133.1019, 119.0840, 105.0700, 91.0537, 81.0712, 67.0543 203.1489 [MþH2O]þ, 159.1133 [MþH2H2O2C3H6]þ, 137.0959 [MþH2O2C3H4 2C2H2]þ, 131.0840, 91.0547, 79.0536 219.1781 [MþH2H2O]þ, 191.0776, 135.1169 [MþH2H2O2O2C3H6 2C2H2]þ, 107.0889, 91.0540 204.0054, 202.9981 [MþH2HCOOH]þ, 189.9899 [MþH2CO2 2CH3]þ, 171.0259, 159.0245, 115.0527
[M2H2HCOH]2, [M2H2BA]2, [M2H2HCOH2C10H12O4]2, [BA2H]2
300.0307 [M2H2CH3]2, 255.0302, 229.0530, 177.0236, 165.0185, 151.0072, 121.0286
301.2204 [M2H22CO]2, 273.2249 [M2H23CO]2, 243.1778, 213.1303, 174.0336, 136.0176
205.1949/20.7 29.62 59
247.0245/20.5 28.29 58
233.0086/21.3 27.64 57
TR, retention time; BA, benzoic acid; GA, gallic acid; TP, Typhae Pollen; PR, Paeoniae Radix Rubra; CR, Curcumae Radix; GR, Gastrodiae Rhizoma; RR, Rhapontici Radix. a Confirmation in comparison with authentic standards. b [M]þ for choline.
CR,RR b-Elemene C15H24
RR Arctinone b C13H10OS2
RR Arctinal C12H8OS2
CR (þ)-Curzeone C15H16O2
213.0915 [MþH2O]þ, 167.0846, 153.0693 [MþH2O2H2O2C3H6]þ, 128.0610 205.0160 [MþH2CO]þ, 202.9972 [MþH2HCOH]þ, 189.9879 [MþH2CO2CH3]þ, 171.0255, 127.0540 205.0096 [MþH2C2H2O]þ, 189.9995 [MþH2C2H2O2CH3]þ, 171.0264 149.1318, 121.1010 [MþH22C3H6]þ, 105.0691, 93.0687 [MþH22C3H6 2C2H4]þ, 79.0550 229.1227/1.6 25.20 56
their MS/MS spectra, losses of glycosyl moieties in both positive and negative ion modes could be generally observed, as well as the fragment ions due to the retro Diels –Alder (RDA) fragmentation pathways at m/z 179, 153, 137 and 123. Peaks 23 and 26 were, respectively, identified as quercetin3-O-neohesperidoside and quercetin-3-glucoside for having the same chromatographic and mass spectral properties with the corresponding reference standards. Peak 21 processed [MþH]þ ions at m/z 757.2179 (C33H40O20), and was 146 Da more than that of Peak 23. Fragment ions at m/z 465.1064 and 303.0498 were obtained by the losses of a rhamnose and a rhamnosylglucose, respectively. By examining the known constituents in Pollen Typhae (14), Peak 21 was tentatively identified as quercetin-3-O-(2G-a-L-rhamnosyl)-rutinoside. Peak 28 displayed the UV lmax at 259, 356 nm, [MþH]þ ions at m/z 595.1652 (C27H30O15), and it was identified as kaempferol-3-O-neohesperidoside by the comparison of the retention time, UV data and MS2 fragmentation patterns with its reference standard. Peak 24 gave the UV lmax at 254, 365 nm, [MþH]þ ions at m/z 741.2221 (C33H40O19), and was 146 Da more than that of Peak 28. Fragment ions at m/z 595.1567, 449.1046 and 287.0540 were obtained by successive losses of a rhamnose, two rhamnose moieties and a glucose residue, respectively. Therefore, Peak 24 was tentatively identified as kaempferol-3-O-(2G-a-L-rhamnosyl)-rutinoside, which had been reported in the pollen of T. angustifolia L (15). Peaks 30 and 33 have the same molecular formula of C28H32O16 and [MþH]þ ions at m/z 625.1757. Both of the two peaks presented high intensity product ions at m/z 479.1187 and 317.0658, indicating the losses of a rhamnose and a rhamnosylglucose moiety. Peak 30 was undoubtedly identified as isorhamnetin-3-O-neohesperidoside by comparing the retention time, UV absorptions and MS/MS spectrum with the reference standard, and Peak 33 was identified as isorhamnetin-3O-rutinoside in accordance with the literature data. Peaks 25 and 34 showed the [MþH]þ ions at m/z 771.2341 (C34H42O20) and 479.1179 (C22H22O12), which had the mass differences of plus or minus 146 Da compared to that of peak 30. They both had similar fragmentation pathways to Peak 30 and obtained aglycone ions at m/z 317.0651 and 317.0659, respectively. Peak 25 was identified as typhaneoside by comparison with the reference standard. And peak 34 could be tentatively identified as and isorhamnetin-3-O-b-glucopyranoside, which was one rhamnose group (146 Da) fewer than peak 30. Peak 32 gave the UV lmax at 225, 339 nm, [MþH]þ ions at m/z 435.1285 (C21H22O10), and produced an aglycone ion at m/z 273.0755 by the loss of a glucose residue. By comparing with ref. (16), Peak 32 was plausibly characterized as isosalipurposide. Peak 44 was identified as apigenin by comparing the chromatographic and mass spectral properties with the reference standard. Peak 36 gave [MþH]þ ions at m/z 447.0926 (C21H18O11), and was 176 Da (C6H10O7) more than that of Peak 44. And, it obtained an aglycone ion at m/z 271.0603, indicating the presence of a glucuronide moiety. Thus, Peak 36 was tentatively identified as apigenin 7-O-b-glucuronide. Peak 37 gave a positively charged molecular ion [MþH]þ at m/z 493.1336 (C23H24O12) and fragment ion at m/z 331.0823 by the loss of a glucose molecule. Thus, Peak 37 was tentatively identified as quercetin-3,30 -dimethyl ether-40 -glucoside, which was reported in T. angustifolia (17). Peaks 40, 42 and 46 were unequivocally Rapid Identification and Simultaneous Quantification of Multiple Constituents in NSTC 893
identified as calycosin, naringenin and isorhamnetin by comparing with the reference standards. Peak 13 showed [M2H]2 ions at m/z 289.0725 (C15H14O6), and MS/MS spectrum showed fragment ions at m/z 245.0844 ([M2H2CO2]2) and 203.0732 ([M2H2CO2 2C2H2O]2). Peak 13 was identified as catechin by comparing those of its reference standard. Peak 11 obtained [M2H]2 ions at m/z 451.1252, and was 162 Da (C6H10O5) more than that of Peak 13. It produced a high intensity [M2H2Glc]2 at m/z 289.0753, and similar fragment ions with Peak 13 at m/z 245.0847 ([M2H2Glc2CO2]2) and 203.0726 ([M2H2Glc2CO2 2C2H2O]2). Thus, Peak 11 was tentatively identified as catechin-7-O-glucoside, which was reported in Paeoniae Radix (18).
Identification of sesquiterpenoids Sesquiterpenoids are unique components in Curcuma (4) and have been reported to show the analgesic and antimicrobial bioactivities (19). A total of 11 sesquiterpenoids were identified in NSTC. Positive ion mode detection is more sensitive for these identified sesquiterpenoid compounds. The neutral loss of one water molecule was generally observed in their MS/MS spectra, even though there were no hydroxyl moieties in the structure. This indicated that the deoxygenation of lactone group and carbonyl group was probably through the fission of C–O bonds and the combination of hydrogen from the nearby position to form a water molecule. Product ions yielded by the losses of a propylene moiety (42 Da) and an ethylene molecule (28 Da) could also be commonly obtained. Comparing with both the reference standard and literature data (20) led to the identification of Peak 53 as germacrone. The [MþH]þ focused on m/z 219.1744 (C15H22O), and product ions were observed at m/z 203.1489 by the loss of one oxygen atom. Other product ions focused on m/z 159.1133 ([MþH2H2O2C3H6]þ) and 137.0959 ([MþH2O2C3H42C2H2]þ). Peak 52 obtained [MþH]þ at m/z 235.1693 (C15H22O2), and was 16 Da (O) more than that of Peak 53. It produced product ions at m/z 217.1583, 199.1508 and 157.1001 by successive losses of one water molecule, two water molecules and a propylene moiety, respectively. Peak 52 was tentatively identified as 13-hydroxygermacrone, which has been reported in the literature (20). Peak 51 showed [MþH]þ at m/z 219.1742 (C15H22O), the same with that of Peak 53. Its product ions focused on m/z 177.1268, 135.1215 and 107.0864 by successive losses of one propylene, one ketene (42 Da) and one ethylene molecule, respectively. Peak 51 was tentatively identified as b-turmerone, which has been reported in the literature (21). Peaks 41 and 54 gave the same [MþH]þ ions at m/z 237.1848. Peak 41 showed product ions at m/z 219.1824 ([MþH2H2O]þ), 135.1128 ([MþH2O2C3H6 2C2H2]þ), and Peak 32 generated similar product ions at m/z 219.1781 ([MþH2H2O]þ), 135.1169 ([MþH2O2C3H6 2C2H2]þ). Based on the literature data (20, 22), Peaks 41 and 54 were tentatively identified as curdione and curcumol, respectively. Peaks 47 and 48 have the same [MþH]þ ions at m/z 231.1378 and the predicted molecular formula was C15H18O2. Both of them displayed major fragment ions at m/z 203.1448 and 185.1334 by successive losses of an ethylene and a water molecule, respectively. According to their retention times in reverse-phase HPLC reported before (23), Peaks 47 and 48 were plausibly identified as epicurzerenone and curzerenone, respectively. 894 Li et al.
Peak 50 had [MþH]þ ions at m/z 217.1586 (C15H20O) and produced fragment ions at m/z 131.0850 and 105.0687 by successive losses of one water, one propylene and one ethylene molecule. Therefore, Peak 50 was tentatively assigned as curzerene (24). Peak 56 obtained [MþH]þ at m/z 229.1227 (C15H16O2) and its fragment ions focused on m/z 213.0915 ([MþH2O]þ) and 153.0693 ([MþH2O2H2O2C3H6]þ). Peak 56 was tentatively identified as curzeone based on the literature information (20). Peak 59 showed [MþH]þ ions and [MþH22C3H6]þ ions at m/z 205.1949 and 121.1010, respectively. According to the literature information reported (22), Peak 59 was tentatively identified as b-elemene. Peak 45 showed [MþH]þ at m/z 265.1436 (C15H20O4) and obtained product ions at m/z 247.1319, 229.1226 and 201.1265 by the progressive losses of one water molecule, two water molecules and a carbon monoxide, respectively. Therefore, Peak 45 was tentatively identified as zedoarofuran (25).
Identification of ketosteroids Peak 29 exhibited the UV lmax at 259, 356 nm, [MþH]þ ions at m/z 481.3154 (C27H44O7) and product ions by a series of losses of one, two, three and four water molecules. The comparison of retention time, UV absorption and MS spectrum with the authentic standard confirmed its identification as b-ecdysterone. Peak 38 gave the same [MþH]þ ions with Peak 29 at m/z 481.3155. It produced product ions at m/z 463.3067, 445.2959, 427.2846 and 409.2940 by the similar fragmentation patterns to Peak 29. Thus, Peak 38 was tentatively identified as an isomer of ecdysterone. Peak 49 gave [MþH]þ ions at m/z 359.2219 (C22H30O4), and also formed fragments by successive losses of one or two H2O moieties. Therefore, it was tentatively identified as palbinone, which has been reported in Paeonia genus (26).
Identification of thiophenes Peaks 55, 57 and 58 exhibited [MþH]þ ions at m/z 249.0038 (C12H8O2S2), 233.0086 (C12H8OS2) and 247.0245 (C13H10OS2), respectively. All of their MS/MS spectra obtained major fragment ions at m/z 189.9899 and 171.0259 (Figure 3). Other product ions of Peaks 55, 57 and 58 focused on m/z 202.9981 ([MþH2HCOOH]þ), 202.9972 ([MþH2HCOH]þ) and 205.0096 ([MþH2C2H2O]þ), respectively. By comparing their quasi-molecular weights and chromatographic properties with the reported compounds in R. uniflorum (27), Peaks 55, 57 and 58 were tentatively identified as arctic acid, arctinal and arctinone b, respectively.
Identification of organic acids and derivatives By comparing the retention time, UV data and MS/MS fragmentation pattern with the reference standards, Peaks 6, 7, 9, 14, 15 and 19 were identified as gallic acid, gastrodin, protocatechuic acid, vanillic acid, chlorogenic acid and syringic acid, respectively. Peak 10 gave the maximum UV absorption at 256, 330 nm, the [M2H]2 ions at m/z 315.0727 (C13H16O9) and was 162 Da more than that of Peak 9. It obtained a high intensity fragment ion at m/z 153.0206 by the loss of a glucose (162 Da) residue. Thus, Peak 10 was identified as protocatechuic acid-3-Oglucoside (28).
Figure 3. Spectra of fragment ions in the analysis of arctic acid (A), arctinal (B) and arctinone b (C) in the positive ion mode.
Peak 35 had the [M2H]2 ions at m/z 273.0777 (C15H14O5) and product ion at m/z 167.0355 by the loss of one C7H6O residue. Thus, Peak 35 was identified as 5-methoxy-2-(3-methoxyphenoxy) benzoic acid. Peaks 22 and 31 showed [M2H]2 ions at m/z 787.0996 (C34H28O22) and 939.1110 (C41H32O26) in the negative ion mode, respectively. Both of them produced fragment ions by losses of one or two gallic acid molecules, suggesting the presence of galloyl group. In accordance with the existing study (29), Peaks 22 and 31 were tentatively identified as 2,3,4,6-tetra-O-galloyl-b-glucose and 1,2,3,4,6-penta-O-galloylb-glucose, respectively.
Identification of alkaloids Alkaloid compounds possess various potential bioactivities such as anti-inflammatory, antimicrobial and anti-asthmatic activities (30). Alkaloids detected in NSTC include amino acids, nucleinic acid and nucleotide. By comparing the retention times and the MS/MS spectra with those of the authentic standards, Peaks 1, 2, 3, 4, 5 and 8 were definitely identified as choline, adenine, L-pyroglutamic acid, adenosine, guanosine, gallic acid and phenylalanine, respectively. In the MS/MS spectra of these nitrogenous compounds, fragment ions yielded by loss of one NH3 moiety could be generally obtained.
Quantification The quantification of five bioactive compounds was determined by ultraviolet absorption at 254 nm for their higher content in the corresponding herbs. Gastrodin, paeoniflorin and b-ecdysterone were selected as the index compounds for the quality control of GR, PR and RR in the compound preparation, respectively. Isorhamnetin-3-O-neohesperidoside and typhaneoside were chosen as the control indexes for TP because of its monarch status in this prescription. The calibration curves with coefficients of correlation (r2) and the data for LOD and LOQ are reported in Table II. The linear range was confirmed according to the content of each compound in NSTC. The LOD and LOQ listed indicated that the limits were low enough to determine these target compounds, which ranged from 0.14 to 1.09 mg/mL and from 0.47 to 3.63 mg/mL, respectively. The RSD value ranges were 0.21–1.33% for intra-day precision and 0.21–1.13% for inter-day precision. The recoveries listed for five compounds were expressed as the average of triplicates + SD at three concentrations, and all recoveries were in the range 96.82–102.12%. As shown by the RSD of 0.33 – 0.85%, samples were stable when kept at 48C within 3 days. All the results indicated that the established method was suitable for the determination of these compounds in NSTC. The contents of five target compounds were analyzed in 10 batches of NSTC, and the data are listed in Table III. Rapid Identification and Simultaneous Quantification of Multiple Constituents in NSTC 895
Table II Method Validation for the Determination of Five Marker Compounds Investigated compounds
Calibration curves
r2 (N ¼ 6)
Linear range (mg/mL)
LOD (mg/mL)
LOQ (mg/mL)
Intra-day RSD of peak area (%) (N ¼ 6)
Inter-day RSD of peak area (%) (N ¼ 3)
Stability RSD within 3 days (%) (N ¼ 3)
Recoveries (average% + SD)
Gastrodin Paeoniflorin Typhaneoside b-Ecdysterone Isorhamnetin-3-O-neohesperidoside
y ¼ 6.597x þ 0.011 y ¼ 20.38x þ 0.3813 y ¼ 158.63x þ 0.0199 y ¼ 148.3x þ 0.0468 y ¼ 206.82x þ 0.0128
0.9996 0.9998 0.9991 0.9996 0.9999
25.5 –510 145 –2900 1.84 –36.8 2.61 –52.2 3.25 –26.2
0.38 1.09 0.15 0.14 0.95
1.26 3.63 0.51 0.47 3.15
1.24 1.33 0.42 0.52 0.21
0.85 1.13 0.34 0.50 0.21
0.76 0.33 0.41 0.85 0.58
96.82 + 5.40 99.45 + 2.31 98.35 + 2.16 98.14 + 2.40 102.12 + 2.65
Table III Mean Concentration (mg/g) of Five Active Compounds in NSTC Products (N ¼ 3) Batch no.
100501
101201
110302
110603
110604
110701
110702
110703
110704
110801
Gastrodin Paeoniflorin b-Ecdysterone Isorhamnetin-3-O-neohesperidoside Typhaneoside
5.80 + 0.01 27.97 + 0.93 0.43 + 0.02 0.24 + 0 0.34 + 0.01
5.12 + 0.10 32.48 + 0.11 0.37 + 0 0.16 + 0 0.20 + 0
3.34 + 0.03 29.89 + 0.61 0.43 + 0.01 0.46 + 0.01 0.57 + 0.02
3.75 + 0.19 29.44 + 0.54 0.47 + 0.1 0.51 + 0.01 0.63 + 0.01
4.16 + 0.33 33.27 + 0.45 0.44 + 0 0.35 + 0.01 0.43 + 0.01
5.61 + 0.04 33.53 + 0 0.44 + 0 0.37 + 0 0.44 + 0.04
5.02 + 0.05 33.46 + 0.48 0.47 + 0 0.40 + 0.01 0.48 + 0
5.57 + 0.09 33.18 + 0.42 0.45 + 0 0.33 + 0 0.41 + 0.01
4.42 + 0.05 36.44 + 0.12 0.55 + 0 0.61 + 0 0.82 + 0.01
5.65 + 0.07 40.45 + 1.31 0.33 + 0.01 0.21 + 0 0.22 + 0.01
Discussion The constituents identified in NSTC could be summarized into several classes such as monoterpene glycosides, flavonoids, sesquiterpenoids, ketosteroids, thiophenes, organic acids and alkaloids according to their structures. By comparing the data of NSTC and its corresponding single raw material, the source of each identified compound was surveyed. As given in Table I, different raw materials possessed their own characteristic chemical contribution, which might cover one or more classes of compounds. For instance, the detected monoterpene glycosides were mainly contributed by PR. Flavonoids were basically originated from TP. The typical compounds in CR were curcuminoids and sesquiterpenoids (4); however, sesquiterpenoids were detected only in NSTC. This would result from the production technology process of NSTC. As shown in Table III, paeoniflorin was found to be the predominant compound, whose content was much higher than other components. Noteworthily, paeoniflorin from the ministerial drug PR was in such a high content, while the contents of the index compounds isorhamnetin-3-O-neohesperidoside and typhaneoside from the monarch drug TP were significantly lower in magnitudes. The results indicated that the content of the monarch drug did not necessarily have to be the highest, but it was still the critical component in quality control and pharmacological studies. What’s more, such combination might produce synergistic effects and reduce possible side effects. This revealed the chemical relation and interaction of NSTC formula, and also reflected one of the main points of the theory of traditional Chinese medicine to some degree. To guarantee the stability, safety and efficacy for clinical use, quality control standard for commercial products of NSTC should be standardized. Conclusion In the present work, a simple and reliable UFLC–DAD –Q-TOF – MS/MS method was established for rapid identification and simultaneous quantification of multiple bioactive constituents in NSTC. A total of 59 constituents including monoterpene glycosides, flavonoids, sesquiterpenoids, ketosteroids, thiophenes, 896 Li et al.
organic acids and alkaloids in the complex matrixes were well separated and identified by comparing the retention times, DAD spectra and TOF/MS data of the analyzed compounds with those of authentic compounds and/or literature data. All of these compounds were also detected in the corresponding single herb. Quantification of five bioactive compounds and analysis of 10 batches commercial NSTC products were carried out, which would be helpful to facilitate its quality control method during production process. What’s more, the identification and structural elucidation of chemical constituents in NSTC also provided essential data for further pharmacological, metabolic and clinical studies.
Acknowledgments We also thank the Teaching Center of Biology Experiment of Sun Yat-Sen University for the instrument support.
Funding This work was supported by grants from the National Science and Technology Major Project (2011ZX09102-011-03).
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Rapid Identification and Simultaneous Quantification of Multiple Constituents in NSTC 897
